The expressed asRNA strategy has mainly been used to study gene function. If the gene in question encodes an essential protein, knockout mutants will be lethal, whereas inducible and titrable downregulation of gene expression by asRNA encoded by a plasmid will enable functional studies. This strategy has been applied for the study of specific genes in a number of different bacteria including E. coli (rpoS) [60], Mycobacterium smegmatis (hisD) [61] and Mycobacterium bovis (ahpC) [62], S. mutans (sgp) [63], S. aureus (srrAB) [64], Clostridium cellulolyticum (cel48F) [65], Thermus thermophilus (cat) [66], Lactobacilllus rhamnosus (welE) [67], Borrelia burgdorferi (ftsZ(Bbu)) [68], Helicobacter pylori (ahpC) [69] and even in the protozoan parasite Entamoeba histolytica (PIG-L) [70]. These studies involved specific genes, but the asRNA expression strategy has also been used for genome-wide studies, in which the bacterial genome is randomly fragmented (~200 to 800 bp) and cloned into vectors. Screening transformants for conditional growth defective phenotypes and characterizing the corresponding expressed antisense fragments has led to the identification of essential genes in S. aureus [71-73] and S. mutans [38] and genes essential to both S. mutans and E. coli [74].

Changing the expression profile of bacteria can have other purposes. Metabolic engineering of bacteria can improve their use as fermenters and cell hosts for recombinant heterologous protein production. For instance, an increase in the butanol-to-acetone ratio in Clostridium acetobutylicum fermentations was obtained using expressed asRNA specific for proteins in the involved pathways [50,75,76]. In another study, asRNA was used to reduce synthesis of a glycosyltransferase, which resulted in a change in the molecular mass of the polysaccharides produced in L. rhamnosus [67]. In E. coli, different strategies have been explored in order to increase recombinant protein production yield, including downregulating the global regulator σ³² to decrease proteolytic degradation of recombinant protein [77], downregulating RNase E to stabilize target product mRNA [78], and reducing endogeneous acetate production to improve heterologous protein synthesis [79] without concomitant growth inhibition. Acetate can cause inhibition of growth and recombinant protein synthesis, but the acetate pathway is also physiologically indispensable, which is why asRNA downregulation of enzymes of the acetate pathway is preferred over gene knockout. Additionally, asRNA expression can be used to protect bacteria used in industrial fermentations against bacteriophages (for reviews see [80-82]).

The expression of asRNA has also been used to validate the point of action of antibiotics. Targeting the proposed molecular target of an antibiotic by asRNA and reducing its expression will sensitize the bacteria to this specific antibiotic. This strategy has especially been used in S. aureus to validate mode of action of antibiotics [72,83] as well as to screen for novel antibacterial agents [36,84-89], but it can also be used to restore antibiotic susceptibility of other resistant strains [90].

Alpha-toxin of S. aureus is well-established as a lethal toxin in mice [91]. A 14- and 16-fold reduction in alpha-toxin expression has been achieved in S. aureus cell cultures by introducing asRNA against the gene encoding this toxin [92,93]. The effect has also been tested in murine models. Mice were infected with the transformed S. aureus strains, and inducer was administered orally [92]. The asRNA expression was shown to eliminate the lethality of the infection. This lead to the proposal of using this strategy to create live-attenuated strains for vaccine candidates [93].

In all the studies mentioned so far, the expressed asRNAs have been complementary to their target mRNAs in an antiparallel orientation. However, expression of parallel asRNA targeting the E. coli lon mRNA has been shown to interfere with target protein synthesis [94] but has not been further investigated.

EGS inhibition of protein synthesis was first investigated in an E. coli plasmid system [99,105]. Alkaline phosphatase and β-galactosidase mRNAs were targeted using single EGSs followed by hammerhead sequences and EGSs covalently linked to M1 RNA. The M1 RNA-EGSs cleaved target mRNA independently of RNase P.

EGS expression has been used for phenotypic conversion of antibiotic-resistant bacteria. Expressing EGSs targeting the resistance-conferring genes of chloramphenicol acetyltransferase (cat), β-lactamase (bla) [99,108] and aminoglycoside 6'-N-acetyltransferase type Ib (aac(6')Ib) [109] restored antibiotic sensitivity, and it was demonstrated that a high EGS-to-target mRNA ratio is required for efficiency [108,110].

Bacterial growth can also be inhibited by targeting essential genes, as e.g. the genes encoding C5 protein subunit of RNase P (rnpA) and subunit A of gyrase (gyrA) in E. coli. Multiple EGSs targeting these to mRNAs showed additive effects and resulted in a 26-fold decrease in growth after EGS induction, the highest growth inhibition achieved in this study in wild-type E. coli [110].

The results obtained in E. coli has been transferred to Salmonella enterica serovar Typhimurium [111]. The non-essential target genes invB and invC express proteins required for type III secretion and for host cell invasion, respectively. EGS targeting of these genes resulted in a decreased host cell invasion rate.

Since the EGSs are expressed from plasmids, the bacteria may try to lose these plasmids to avoid inhibition [109]. Plasmid loss in liquid cultures of bacteria have been shown to begin after 6 hours of growth [108]. In another study transformants containing EGSs with high inhibition index values lost their plasmids more often, exhibited smaller colony size when induced and were more difficult to grow in general than the corresponding low inhibition index EGS transformants [110]. It would seem that the bacteria, when exposed to more effective EGSs, will try harder to escape the growth-inhibiting agents. For in vitro applications, the use of antibiotic resistance markers is routinely used to prevent loss of plasmid, but the EGSs may also be administered directly as chemically synthesized molecules, avoiding the problem of plasmid loss.

Even though the results described here are obtained through induced EGS expression from plasmids transformed into bacteria, the technology can, in principle, be transferred to in vivo applications. Guerrier-Takada et al. suggest altering the so-called R-plasmids of enteric bacteria and derivatives, which are responsible for establishment and transfer of drug resistance in clinical populations, to carry EGS-encoding genes instead of genes for drug resistance [108]. Another option may be to use carriers such as fluid liposomes, which has been shown to facilitate E. coli cell entry of both plasmid DNA and antisense oligonucleotides [112]. Chemically synthesized EGSs could be added instead of plasmid DNA, and the EGSs could be DNA-based instead of RNA-based to increase nuclease stability. However, DNA-based EGSs have been shown to be 10-fold less efficient in vitro than unmodified RNA-based EGSs [113].

Using EGS technology for microbial antisense inhibition still needs further development and optimization, and some of the modifications investigated in human cells are still to be investigated in bacterial cells. It should be kept in mind, though, that due to differences in the structural requirements of EGSs directing mRNA cleavage by either human or bacterial RNase P, the results obtained in one system may not be transferred to the other [99].

Replacing one of the non-bridging oxygens of the phosphodiester bonds of DNA with sulfur gives phosphorothioate oligodeoxynucleotides (PS-ODNs, Fig. 9), which are some of the most studied oligonucleotides. As for the MPOs, the introduction of sulfur atoms introduces chirality at the phosphorus centers, and the PS-ODNs behave differently depending on the diastereomeric configuration of the linkages. Thus, the all-Rp version has a higher binding affinity and a greater RNase H activation but a lower in vitro stability against nucleases than the all-Sp diastereomer. PS-ODNs of mixed diastereomeric linkages show intermediate abilities [163,164]. It has been indicated that nuclease stability is one of the most important factors for PS-ODN efficacy [165], and hence the stereocontrolled synthesis of antisense PS-ODNs has been well investigated (for a review see [163]).

The PS-ODNs are highly soluble, and their mechanism of action involves binding to target RNA and activating RNase H for cleavage of the target (Fig. 1). RNase H-dependent ODNs can be targeted to virtually any region of the RNA or mRNA, whereas antisense agents inhibiting expression through steric blocking should be targeted to the 5' end or the initiation codon region [138]. The phosphorothioate DNA analogues can display poor sequence specificity, since even short duplexes of PS-ODN/RNA can be degraded by RNase H, and PS-ODNs can therefore induce cleavage of sequences with only partial homology to the targeted RNA [166]. They have also been shown to induce sequence-independent effects by interacting with cellular proteins, though this is suggested to be synergistic with the downregulating effect of the PS-ODN [138].

PS-ODNs have been studied and used extensively in eukaryotic systems, and this has led to the development of the first FDA-approved antisense drug, fomivirsen [167] (commercially known as Vitravene). The 21-mer PS-ODN (Fig. 10) designated ISIS-2922 targets the major immediate-early gene of human cytomegalovirus in HIV-patients with retinitis. More PS-ODN drugs for treatment of human diseases are in clinical trials, but the use of PS-ODNs in bacteria has been rather limited and nearly confined to Mycobacteria (Table 1).

Applying PS-ODN technology to M. tuberculosis demonstrated several important parameters:

• concentrations of ≥10 μM were most effective (much higher concentrations are undesirable due to increase in non-sequence-specific interactions with host proteins and nucleic acids [168]),

• PS-ODNs should be at least 18 and preferably 24 bases in length to minimize random hybridization,

• and mRNA targets should have a low propensity to form stable secondary structure at 37°C (based on a secondary structure analysis program) [168,169].

This was elucidated from the targeting of three different sites in glnA1 mRNA encoding glutamine synthetase, the export of which is associated with pathogenicity and formation of cell wall structure [170]. Besides glnA1 [168], PS-ODNs have been used to target

• fbpA, -B and -C [169,171] (single operon genes encoding three proteins of the 30/32-kDa protein complex that are essential for mycobacterial cell wall synthesis and leading tuberculosis vaccine candidates [172]),

• alr [169] (encoding alanine racemase essential for synthesis of cell wall peptidoglycan)

• and ino1 [173] (encoding inositol-1-phosphate synthase, a key enzyme in phosphatidyl-inositol synthesis).

Combinations of PS-ODNs targeting these mRNAs demonstrated growth inhibition of M. tuberculosis and sensitized the cells to conventional drugs. Furthermore, it was most importantly demonstrated that bacterial growth could be inhibited in human macrophages infected with M. tuberculosis, albeit with a fairly low efficiency [171].

PS-ODNs have been used in a few other bacterial species. The MecR1-mecI gene of S. aureus regulates synthesis of penicillin-binding protein 2a (PBP2a), which mediates resistance to methicillin and all β-lactam antibiotics. A PS-ODN targeting MecR1 mRNA restored antibiotic susceptibility of a methicillin-resistant S. aureus (MRSA) strain [174].

Another example of restoration of antibiotic susceptibility comes from mutant E. coli strains, in which a PS-ODN restored a norfloxacin sensitive phenotype, although with lower efficiency [175].

In Streptococcus mutans, gtfB encodes glycosyltransferase, which is responsible for synthesis of water-insoluble glucans facilitating adhesion of the organism to the surface of teeth. The GtfB synthesis and activity as well as biofilm formation of S. mutans was inhibited by an anti-gtfB PS-ODN, but bacterial growth was unaffected [176]. Nevertheless, this might be useful in caries prevention.

In this study, uptake of PS-ODN and hence inhibitory efficiency in this Gram-positive bacterium was improved by adding a transfection reagent of cationic polymers, though inhibition was observed with free PS-ODN as well [176].

Mycobacteria are neither truly Gram-negative nor -positive [177]. Their outer lipid bilayer is the thickest biological membrane hitherto known, and the exceptionally low permeability of this membrane renders mycobacteria resistant to many antibiotics [178]. The ability of lipophilic drugs to solubilize within the lipid portion of the outer wall layer leads to improved efficiency over hydrophilic drugs, which are prevented from traversing the cell envelope [179]. Li et al. claim that the PS-ODNs should be more easily taken up due to improved lipophilicity [173].

In M. smegmatis, targeting the aspartokinase (ask) gene using PS-ODNs was only effective in the presence of low levels of ethambutol, which increases permeability of the cell wall [180]. As mentioned above, PS-ODNs of different targets have been shown to enter mycobacterial cells unassisted and inhibit growth. However, improvement of uptake is still necessary to further improve the efficiency. Physical methods like heat shock and electroporation have been used as well as mutant strains with a permeable membrane [174,175], but these methods are not suited for targeting wildtype bacteria in tissue. Combining administration of PS-ODNs with conventional antibiotics increasing cell permeability seems fairly straightforward. Different as well as related strategies involve

• covalently attaching PS-ODNs to D-cycloserine (antibiotic thought to be taken up by D-alanine uptake system) or biotin [180,181],

• tethering PS-ODNs to amikacin-moiety (antibiotic targeting 30S ribosomal subunit),

• "softening" the cell wall using subinhibitory concentrations of antibiotics [168],

• incorporation of terminal 2'-O-methyl ribose residues (promotes stability and diminishes animal toxicity),

• 3' addition of a nonhybridizing poly-G tail (postulated to improve uptake in eukaryotes) [169] and

• encapsulation into fluid liposomes [112].

Most of these strategies resulted in only slightly enhanced efficacy if any, so the ultimate uptake-enhancing strategy is yet to be developed.

Peptide nucleic acids (PNAs, Fig. 9) were developed by Nielsen et al. in 1991 [182]. They are nucleic acid analogues, which are based on an aminoethylglycin backbone with acetyl linkers to the nucleobases [183]. This very flexible pseudopeptide backbone is uncharged and thus avoids the electrostatic repulsion, a natural phosphate-ribose backbone would induce during hybridisation. This is thought to be the reason, why these PNAs can form very stable duplexes or triplexes with either single-stranded or double-stranded DNA or RNA [138]. Another consequence of this unnatural backbone is that the PNAs are not degraded by enzymes such as nucleases and proteases [184], and they are also not recognized by RNase H, so the mechanism of action involves binding and steric blocking of ribosomal assembly and translation (Fig. 1). However, being neutral and only slightly hydrophilic molecules, PNAs suffer from low aqueous solubility compared to DNA molecules. Increased solubility can be achieved by extending the PNA sequence with charged amino acid residues such as lysines [185] or by incorporating solubility enhancers (E-linkers) [186] prepared by replacing the nucleobases of the standard PNA monomers with either neutral or positively charged hydrophilic moieties. Though these E-linkers do not influence the hybridization performance of the PNAs, PNA probes with solubility enhancers have been found to be almost incapable of penetrating cell walls of a number of Gram-positive bacteria in whole cell FISH analysis [187], so they should not be used indiscriminately. A different solution has been to grow target cells in 10% LB media to overcome solubility limitations of PNAs [188,189].

A number of different analogues have been produced introducing different non-natural nucleobases into the PNAs [185], and some of these analogues resulted in a high preference for RNA binding over DNA binding [190,191].

PNA oligos are usually very short, since increasing the length generally results in decreased solubility [186], and shorter PNAs are more likely to efficiently enter cells [27]. For antisense applications in bacteria, 9–12-mer PNAs with low self-complementarity and an average GC content are recommended [192]. A low GC content, especially in short PNAs, could result in low binding affinity, whereas a high GC content could cause problems with respect to synthesis and solubility [183].

Triplexes formed between one homopurine DNA or RNA strand (composed of adenine and guanine bases only) and two sequence-complementary PNA strands are extraordinarily stable [183]. One PNA strand binds through Watson-Crick base pairing (preferably in antiparallel orientation) and the other via Hoogsteen base pairing (preferably in parallel orientation). The PNA strands can be advantageously connected to each other covalently by a flexible linker (e.g. three 8-amino-3,6-dioxaoctanoic acid units) to create a bis-PNA (Fig. 11). Furthermore, the binding can be rendered pH independent by replacing cytosines in the Hoogsteen base pairing strand with pseudoisocytosines [193].

For Gram-negative bacteria, the peptidoglycan-layer, the lipid bilayer, and especially the external lipopolysaccharide (LPS) layer provide the bacteria with an efficient first-line of defence and the PNA with a major challenge [194]. PNAs are larger than most drugs (about 2000–4000 MW [195]) and are not readily taken up by bacteria, so different strategies have been developed to facilitate cellular uptake (see reviews by Nielsen [195-197]). Methods for physically disrupting cell membranes are limited to cells in culture, but strategies involving cationic liposomes and conjugation to fatty acids or peptides can be applied in tissues as well. Conjugation of PNAs to small cationic peptides termed cell-penetrating peptides (CPPs) appears to be the most efficient strategy for PNA cell entry [198]. (KFF)₃K, shown by Vaara and Porro to have a cell wall-permeabilizing effect, has become one of the most widely used CPPs [199].

PNA technology has been used to target a number of different E. coli RNAs and genes encoding essential proteins. These targets include the peptidyl transferase center, the α-sarcin loop [189,192] and domain II of 23S rRNA [200], the P15 loop region of RNase P RNA [153], and mRNAs encoding β-galactosidase (lacZ), β-lactamase (bla) [188], and the acyl carrier protein Acp (acpP) [192,201] (Table 2). The obtained results are not directly comparable due to differences in growth medium, starting inoculum etc, but the following will comprise an overview of the development.

Bis-PNAs targeting the peptidyl transferase center, the α-sarcin loop [189], and domain II [200] of 23S rRNA were shown to inhibit translation in a cell-free translation/transcription system. Substantially lower concentrations were required for in vitro inhibition when applying two different anti-bla PNAs and an anti-lacZ PNA [188]. Specific inhibition of the RNase P holoenzyme was also observed at very low concentrations when using a peptide-PNA targeting the P15 loop region of the RNase P RNA. It was shown to bind its target essentially irreversibly in vitro and disrupt local secondary structure in the catalytic core [153].

A rather moderate inhibition of cell growth in culture is a common feature of most of these PNAs, even though they are efficient inhibitors in cell-free systems. This is true at least for wildtype E. coli strains such as K-12 or DH5α. However, the PNAs have a greatly increased inhibitory effect on permeable mutant strains such as AS19 [153,188,189] and SM101 [201], and inefficient uptake and passage through the outer membrane is therefore considered to be the reason for this somewhat moderate cell growth inhibition observed in wildtype. Attachment of the CPP, (KFF)₃K, to PNAs have been shown to improve the inhibitory effect. For instance, the aforementioned anti-α-sarcin loop bis-PNA was in a following study conjugated to (KFF)₃K, and this resulting peptide-PNA exhibited a low minimal inhibitory concentration (MIC) for E. coli K-12. (KFF)₃K was also coupled to an anti-acpP PNA. Here, the inhibitory effect was even greater. In fact, it was recently demonstrated that this peptide-PNA has a lower MIC than the conventional antibiotics ampicillin, chloramphenicol, streptomycin and trimethoprim and is more potent on a molar basis [202]. This study also indicated that peptide-PNAs accumulate in bacteria through rapid uptake and slow passive efflux, and that they mediate a long post-antibiotic effect of more than 11 hours. So even though cell entry is still a challenge, once inside the cells the peptide-PNAs appear to be retained for hours, unlike conventional antibiotics that efflux within minutes [203].

The application of PNAs has been taken a step further. To set up a simple model for the growth of an extracellular pathogen in a host, HeLa cell cultures were infected with non-invasive wildtype E. coli. The anti-acpP peptide-PNA was added immediately post-infection. The PNA was bactericidal at low micromolar concentrations, and no effect was visible for the HeLa cell growth. However, peptide-PNA is still, in animal models, inferior to conventional antibiotics in potency [192].

Another group tested this anti-acpP peptide-PNA in an animal disease model [201]. BALB/c mice were challenged by i.p. injection of a 90% lethal dose (LD₉₀) of E. coli K-12 or LD₇₀ of SM101 mutant strain with a defective outer membrane. Administering anti-acpP peptide-PNA intravenously to the mice rescued up to 60% and 100% of the infected animals, respectively.

The inhibitory effect of peptide-PNAs has recently also been demonstrated for another Gram-negative bacterium, the human pathogen Klebsiella pneumoniae [204]. Targeting essential genes gyrA (DNA gyrase subunit A) and ompA (major outer membrane protein A) resulted in inhibition of protein synthesis and growth but at rather high MICs. Furthermore, the levels of the two transcripts were strongly reduced indicating an antigene effect. This is possible, since PNAs can invade double-stranded DNA and form highly stable triplex-invasion complexes that are strong enough to inhibit gene transcription and regulation. Furthermore, human epithelial fibroblasts (IMR90) were infected with multiresistant K. pneumoniae, and peptide-PNA treatment was bactericidal without visible affect on IMR90 cell growth.

Though Gram-positive bacteria are important human pathogens, they have been given little attention as target candidates for PNAs. Nekhotiaeva et al. recently demonstrated that peptide-PNAs are indeed effective in inhibiting growth of Gram-positive bacterium S. aureus [205]. (KFF)₃K-PNAs were used to target chromosomally encoded alkaline phosphatase (phoB), putative peptidoglycan pentaglycine interpeptide biosynthesis protein (fmhB), gyrase A (gyrA) and HmrB protein (hrmB, which is an ortholog of the E. coli acpP gene). Generally, at least 10 μM concentrations were required for inhibition.

Passage across the outer membrane of Gram-negative bacteria has been indicated to be the rate-limiting step of peptide-PNA uptake [198]. Gram-positive bacteria should in their lack of an outer membrane pose an easier target, but it appears that CPPs are still required for cellular uptake. The mechanism has not yet been elucidated, but 'direct' cell permeation has been suggested as the most likely explanation for peptide-PNA entry into both S. aureus and E. coli [198,205].

As mentioned in section 6.2, the outer lipid bilayer of mycobacteria has an exceptionally low permeability. Nevertheless, peptide-PNAs have been shown to enter mycobacterial cells and inhibit growth by antisense inhibition [206]. (KFF)₃K-PNAs were used to target inhA, which encodes the essential protein enoyl reductase, and M. smegmatis growth was inhibited at fairly low MICs.

Further optimization of peptide-PNAs to increase efficiency is still required. The linker connecting peptide and PNA is one target of optimization [153], since Good et al. [192] showed the linker not to be a 'silent player'.

The cell delivery efficiency of CPPs is another target for optimization. The widely used (KFF)₃K carrier peptide has been found to induce haemolysis in human erythrocytes [199] and histamine release in some mammals [205], so alternative CPPs would be needed for broad medical applications of PNAs. New peptides have been proven efficient in PNA delivery into S. aureus [205] as well as into M. smegmatis [206]. Thus, improvement of carrier peptides should be possible, though these new CPPs still need further characterization.

PNA targeting has not been limited to rRNA and mRNA, also non-coding regulatory RNAs have been used as targets for PNAs [207]. Sok-antisense-RNA inhibits synthesis of Hok protein, which induces host cell killing. Introducing (KFF)₃K-PNAs against plasmid-encoded Sok-antisense-RNA leads to Hok protein synthesis and cell killing in E. coli (for description of hok/sok toxin-antitoxin system see [208]), and the (KFF)₃K-PNA was actually more inhibitory than rifampicin. However, it still remains to be demonstrated that (KFF)₃K-PNA targeting antitoxins can activate suicide in plasmid-free E. coli cells.

Phosphorodiamidate morpholino oligomers (PMOs, Fig. 9) are oligonucleotide mimics, in which the deoxyriboses of DNA have been replaced by morpholine rings coupled by non-ionic phosphorodiamidate intersubunit linkages [209,210]. Despite being uncharged, the PMOs show excellent aqueous solubility, possibly due to good stacking of the bases and shielding of hydrophobic faces [209]. Furthermore, PMOs have displayed resistance to a variety of nucleases, proteases, esterases, and degradative enzymes in serum and liver homogenates [211], and like PNAs their mechanism of action is RNase H-independent and involves sequence-specific binding and steric blocking of ribosomal assembly and hence translation (Fig. 1). However, the binding affinity of PMOs is lower than for the equivalent PNAs [212].

PMO-mediated translation inhibition in bacteria has mainly been focused on targeting the mRNA encoding the essential acyl carrier protein Acp (acpP) of E. coli (Table 3), though the effects of PMOs on Bacillus anthracis are currently being studied [213]. As for PNAs, the outer membrane of the Gram-negative E. coli poses a barrier for PMO cell uptake, and 10–20-mer PMOs were only shown to be effective in mutants with a defective outer membrane [212,214]. A specific 11-mer anti-acpP PMO did, however, briefly inhibit growth of wild-type E. coli [215].

To improve PMO efficiency, conjugation to CPPs was tested. (KFF)₃KC, resembling the (KFF)₃K peptide used to promote cell uptake of PNAs (see section 6.2), was effective in facilitating cell uptake of 20-mer PMOs [214]. The free peptide was shown to be toxic at 20 μM, but conjugation to the PMO apparently eliminated this toxicity.

Using the 11-mer anti-acpP PMO, another three CPPs were shown to efficiently assist in transporting the PMO into cells [216]. In this study, pathogenic strains of E. coli (enteropathogenic E. coli [EPEC]) and S. enterica serovar Typhimurium appeared more susceptible to peptide-PMO inhibition than wildtype E. coli, possibly due to different extents of peptide hydrolysis in the different strains. EPEC growth in tissue culture could also be inhibited by the peptide-PMOs, but their efficiency in reducing bacterial viability was orders of magnitude greater in tissue culture than in liquid culture. This may be due to differences in bacterial growth in the two types of culture.

The inhibitory effect, though modest, of the anti-acpP PMO in a mouse model of E. coli peritonitis was the first demonstration of antisense DNA analogues inhibiting bacterial growth in an animal model [215]. The conjugate of this PMO and (RFF)₃RXB-peptide was shown to be about 50–100 times more potent than the single PMO in the mouse model [217]. In the mouse model of E. coli peritonitis, the anti-acpP peptide-PMO was found to reduce the number of colony-forming units (CFU) in the blood and promote survival at a potency at least 15 times greater than ampicillin. Though the results suggested high doses of the conjugate to be toxic, preliminary toxicology data on treatment with about 20 times higher doses of anti-acpP peptide-PMO showed no apparent toxicity [217]. Since the possible toxicity is thought to be caused by the peptide moiety and not the PMO (PMO compounds are considered safe in humans [218]), further optimizations on this part of the compound will be necessary, unless the toxicological studies can acquit this peptide of toxicity.

The therapeutic use of antisense PMOs seem highly possible, and further pharmokinetic and toxicological studies will help improve this compound for antibacterial treatment.