WO2001027262A1

WO2001027262A1 - Gene selection using pnas

Info

Publication number: WO2001027262A1
Application number: PCT/DK2000/000581
Authority: WO
Inventors: Peter E. Nielsen; Carsten Schou; Margit Wissenbach
Original assignee: Pantheco A/S
Priority date: 1999-10-13
Filing date: 2000-10-13
Publication date: 2001-04-19
Also published as: EP1220903A1; JP2003511068A; AU7773100A

Abstract

The invention concerns novel drugs for use in combating infectious micro-organisms, in particular bacteria. More particular the invention concerns peptide nucleic acid (PNA) sequences which are modified by conjugating cationic peptides to the PNA in order to obtain novel PHA molecules with enhanced anti-infective properties. The PNA sequences ae used in a method for identifying one or more target genes in a micro-organism such as E.coli, for developing an anti-infective treatment.

Description

GENE SELECTION USING PNAS

The present invention concerns novel drugs for use in combating infectious micro-organisms, in particular bacteria. More particular the invention concerns peptide nucleic acid (PNA) sequences which are selected to be effective in combating micro-organisms and modified in order to obtain novel PNA molecules with enhanced anti- infective properties.

BACKGROUND OF THE INVENTION

From the discovery of penicillin in the 1940' s there has been an ever growing search for new drugs. Many drugs or antibiotics have been discovered or developed from already existing drugs. However, over the years many strains of bacteria have become resistant to one or more of the currently available drugs which were effective drugs in the past. The number of antibiotic drugs currently being used by clinicians are more than 100.

Most antibiotics are products of natural microbic populations and resistant traits found in these populations can disseminate between species and appear to have been acquired by pathogens under selective pressure from antibiotics used in agriculture and medicine (Davis

1994) . Antibiotic resistance may be generated in bacteria harbouring genes that encode enzymes that either chemically alter or degrade the antibiotics. Another possibility is that the bacteria encodes enzymes that makes the cell wall impervious to antibiotics or encode efflux pumps that eject antibiotics from the cells before they can exert their effects (Levy 1998) . Because of the emergence of antibiotic resistant bacterial pathogens, there is an on-going need for new therapeutic strategies. One strategy to avoid problems caused by resistance genes is to develop anti-infective drugs from novel chemical classes -for which specific resistance traits do not exist.

Antisense agents offer a novel strategy in combating diseases, as well as opportunities to employ new chemical classes in the drug design.

Oligonucleotides can interact with native DNA and RNA in several ways. One of these is duplex formation between an oligonucleotide and a single stranded nucleic acid. Another is triplex formation between an oligonucleotide and double stranded DNA to form a triplex structure.

Results from basic research have been encouraging, and antisense oligonucleotide drug formulations against viral and disease causing human genes are progressing through clinical trials. Efficient antisense inhibition of bacterial genes also could have wide applications, however, there have been few attempts to extend antisense technology to bacteria.

Peptide nucleic acids (PNA) are compounds that in certain respects are similar to oligonucleotides and their analogs and thus may mimic DNA and RNA. In PNA, ^~he deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991) (Fig. 1). Each subunit, or monomer, has a naturally occurring or non naturally occurring nucleobase attached to this backbone. One such backbone is constructed of repeating units of N- (2-aminoethyl) glycine linked through amide bonds. PNA hybridises with complementary nucleic acids through Watson and Crick base pairing and helix formation (Egholm et al . 1993). The Pseudo-peptide backbone provides superior hybridization properties (Egholm et al . 1993), resistance to enzymatic degradation (Demidov et al . 1994) and access to a variety of chemical modifications (Nielsen and Haai a 1997) .

PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm' s . This high thermal stability might be attributed to the lack of ' charge repulsion due to the neutral backbone in PNA. In addition to increased affinity, PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex, there is seen an 8 to 20°C drop in the Tm.

Furthermore, homopyrimidine PNA oligomers form extremely stable PNA₂-DNA triplexes with sequence complementary targets in DNA or RNA oligomers. Finally, PNAs may bind to double stranded DNA or RNA by helix invasion.

An advantage of PNA compared to oligonucleotides is that their polyamide backbones (having appropriate nucleobases or other side chain groups attached thereto) is not recognised by either nucleases or proteases and are not cleaved. As a result, PNAs are resistant to degradation by enzymes unlike nucleic acids and peptides.

For antisense application, target bound PNA can cause steric hindrance of DNA ad RNA polymerases, reverse transcription, telomerase and the ribosomes (Hanvey et al. 1992, Knudsen et a. 1996, Good and Nielsen 1998), etc.

A general difficulty when using antisense agents is cell uptake. A variety of strategies to improve uptake can be envisioned and there are reports of improved uptake into eukaryotic cells using lipids (Lewis et al. 1996), encapsulation (Meyer et al. 1998) and carrier strategies (Nyce and Metzger 1997, Pooga et al, 1998) .

WO 99/05302 discloses a PNA conjugate consisting of PNA and the transporter peptide transportan, which peptide may be used for transport cross a Iipid membrane and for delivery of the PNA into interactive contact with intracellular polynucleotides.

US-A-5 777 078 discloses a pore-forming compound which comprises a delivery agent recognising the target cell and being linked to a pore-forming agent, such as a bacterial exotoxin. The compound is administered together with a drug such as PNA.

As an antisense agent for micro-organisms, PNA may have unique advantages. It has been demonstrated that PNA based antisense agents for bacterial application can control cell growth and growth phenotypes when targeted to Escherichia coli rRNA and mRNA (Good and Nielsen

1998a, b and WO 99/13893) .

However, none of these disclosures discuss ways of obtaining highly effective PNA sequences.

Furthermore, for bacterial application, poor uptake is expected, because bacteria have stringent barriers against foreign molecules and antisense oligomer containing nucleobases appear to be too large for efficient uptake. The results obtained by Good and Nielsen ( 1998a, b) indicate that PNA oligomers enter bacterial cells poorly by passive diffusion across the Iipid bilayers. US-A-5 834 430 discloses the use of potentiating agents, such as short cationic peptides in the potentiation of antibiotics. The agent and the antibiotic are co- administered.

WO 96/11205 disclose PNA conjugates, wherein a conjugated moiety may be placed on terminal or nonterminal parts of the backbone of PNA in order to functionalise the PNA. The conjugated moieties may be reporter enzymes or molecules, steroids, carbohydrate, terpenes, peptides, proteins, etc. It is suggested that the conjugates among other properties may possess improved transfer properties for crossing cellular membranes. However, WO 96/11205 does not disclose conjugates which may cross bacterial membranes .

WO 98/52614 discloses a method of enhancing transport over biological membranes, e.g. a bacterial cell wall. According to this publication, biological active agents such as PNA may be conjugated to a transporter polymer in order to enhance the transmembrane transport. The transporter polymer consists of 6-25 subunits, at least 50% of which contain a guanidino or amidino sidechain moity and wherein at least 6 contiguous subunits contain guanidino and/or amidino sidechains. A preferred transporter polymer is a polypeptide containing 9 arginine. However, WO 98/52614 does not disclose any PNA sequences which may target bacterial genes.

Thus, despite the promising results in the use of the PNA technology obtained previously, there is a great need of developing new PNA antisense drugs which are effective in combating micro-organisms. SUMMARY OF THE INVENTION

The present invention concerns a new strategy for combating bacteria. It has previously been shown that antisense PNA can inhibit growth of bacteria. However, a slow diffusion of the PNA over the bacterial cell wall combined with less effective target sequences, a practical application of the PNA as an antibiotic has not been possible previously. According to the present invention, a practical application in tolerable concentration may be achieved by selecting the right gene or genes as target and combining a PNA sequence targeting such gene or genes with a peptide or peptide-like sequence which enhances the efficiency of the transport over the cell membrane.

In a first aspect of the present invention, the present invention concerns a method of identifying specific advantageous antisense PNA sequences which may be used in combating micro-organisms.

Accordingly, the present invention concerns a method of identifying one or more target genes in a micro-organism, which target gene(s) may be the basis for an anti- infective treatment, comprising:

a) selecting potential target genes known to be present in the micro-organism,

b) obtaining one or more complementary (antisense) PNA sequences for each of the selected potential target genes,

c) mixing one or more antisense PNA sequences separately or combined with the micro-organism in a growth media, d) monitoring the growth of the micro-organism in the presence and absence of antisense PNA, and

e) identifying a target gene as a gene which blockage by the corresponding antisense PNA results in a restricted growth of the micro-organism compared to the growth in the absence of antisense PNA.

In a preferred embodiment, the method comprises the use of a PNA sequence linked to an activity enhancing moiety. More preferred, the activity enhancing moiety is a peptide which enhances the access of the PNA to the gene target in the micro-organism, for example by enhances the crossing of the PNA over the cell wall of the microorganism.

Surprisingly, it has been found out that by incorporating a cationic peptide, an enhanced anti-infective effect can be observed. The important feature of such a modified PNA molecules seems to be a pattern comprising positively charged and lipophilic amino acids or amino acid analogs. An anti-infective effect is found with different orientation of the peptide in relation to the PNA- sequence.

In a preferred method, the peptide is (Lys-Phe-Phe) ₃.

Another aspect of the invention concerns a PNA molecule comprising a PNA sequence which is complementary (antisense) to at least a part of a target gene in a micro-organism, which target gene is identifiable according to the method defined in the first aspect of the invention as stated above.

The PNA molecule may further comprise an activity enhancing moiety such as a cationic peptide.

In yet another aspect of the invention, the PNA molecules are used in the manufacture of medicaments for the treatment or prevention of infectious diseases or for disinfecting non-living objects.

In a further aspect, the invention concerns a composition for treating or preventing infectious diseases or disinfecting non-living objects.

In yet a further aspect, the invention concerns the treatment or prevention of infectious diseases or treatment of non-living objects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the chemical structure of DNA and PNA oligomers.

FIGURE 2 shows the principle in conjugation using smcc

FIGURE 3 shows the nucleotide sequence of the mrcA (ponA) gene encoding PBP1A. The sequence of the gene (accession number X02164) was obtained from the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al . 1985, Eur J Biochem 147:437-46) . Two possible start codons have been identified (highlighted) . Bases 1-2688 are shown (ending with stop codon) .

FIGURE 4 shows the nucleotide sequence of the mrdA gene encoding PBP2. The sequence (accession number AE000168, bases 4051-5952, numbered 1-2000) was obtained from the E. coli genome database at the NCBI (Genbank, National Centre for Biotechnology Information, USA) . The start codon is highlighted.

FIGURE 5 shows the inhibitory effect exhibited by the PNA specificity PNA 109. The figure shows bacterial growth curves obtained without PNA-peptide and with PNA-peptide present at final concentrations varying from 200 nM to 1000 nM.

FIGURE 6 shows the inhibitory effect exhibited by the PNA specificity PNA 111. The figure shows bacterial growth curves obtained without PNA-peptide and with PNA-peptide present at final concentrations varying from 200 nM to 1000 nM.

DETAILED DESCRIPTION OF THE INVENTION

Antisense PNAs can inhibit bacterial gene expression with gene and sequence specificity (Good and Nielsen 1998a, b and WO 99/13893) . The approach may prove practical as a tool for functional genomics and as a source for novel antimicrobial drugs. However, improvements on standard PNA are required to increase antisense potencies. One limit to activity appears to be the selection of the right target gene. Another limit, cellular entry. Bacteria effectively exclude the entry of large molecular weight foreign compounds, and previous results for in vitro and cellular assays seem to show that the cell barrier restricts antisense effects. Accordingly, the present invention concerns strategies select target genes and improve the activity of antisense potencies.

Without being bound by theory, it is believed that short cationic peptides may lead to an improved PNA uptake over the bacterial cell wall. It is believed that the short peptides act by penetrating the cell wall, allowing the modified PNA molecule to cross the cell wall and membrane to get access to structures inside the cell, such as the genome, mRNAs, the ribosome, etc. However, an improved accessibility to the nucleic acid target or an improved binding of the PNA may also add to the overall effect observed.

However, an improved accessibility is not enough in providing an effective anti-infective PNA molecule. A highly important feature is the access to the right PNA sequence, which depends on an identification of gene targets which are essential for the growth of the host micro-organism.

According to the invention, PNA molecules may be synthesised, which molecules may be used in a specific and efficient inhibition of bacterial genes with nanomolar concentrations. Antisense potencies in this concentration are consistent with practical applications of the technology. Thus, the present invention has made it possible to administer PNA in an efficient concentrations which is also acceptable to the patient.

Accordingly, the present invention concerns method of identifying one or more target genes in a micro-organism, which target gene(s) may be the basis for an anti- infective treatment, comprising:

a) selecting potential target genes known to be present in the micro-organism,

c) mixing one or more antisense PNA sequences separately or combined with the micro-organism in a growth media,

d) monitoring the growth of the micro-organism in the presence and absence of antisense PNA, and

The selection of potential gene targets and testing of ensuing PNA constructs have been performed with E. coli as an example of selection of gene targets in a microorganism. However, it is clear that the principle can be applied to any other micro-organisms, especially to the group of bacteria.

The organism Escherichia coli K-12 MG1655 was obtained from the E. coli Genetic Stock Center at Yale University, Ct, USA. The genome of the organism has been fully sequenced and includes a total of 4.639.221 bp and 4289 open reading frames.

Potential target genes were retrieved from the complete E. coli genome at Genbank. Target sequences with a length of 12 bases were selected around the start codon region of each open reading frame. The presence of homologous gene and target sequences in bacterial genomes and the human genome were analyzed by using the BLAST 2.0 programs at the NCBI (Genbank, National Center for Biotechnology Information) www BLAST server.

In another aspect of the present invention, the target genes may be use to identify compounds which may be used to inhibit growth of micro-organisms, such as bacteria. Definition of selection criteria

The following general considerations could be followed when selecting a potential gene target:

1. Spectrum of activity:

A broad spectrum antibiotic can often be used immediately without a detailed diagnosis; there is a good probability to hit the pathogen, but on the other hand, the resident microflora may also be affected, thus increasing the chance for new pathogens to grow up after treatment. For these and resistance related reasons, it may be advantageous to aim at designing PNA antimicrobials with a restricted spectrum.

2. Bactericidal effect:

For patients with a normal immune status bacteriostatic antimicrobial agents are sufficient in many cases. The static effect gives the immune system time to catch up with the invader and thus do the rest of the job. However, for weak or immunocompromised patients a bacteriostatic effect is not sufficient. It is therefore further advantageous to design bactericidal PNA antimicrobials .

3. Selective toxicity:

The antimicrobial PNA constructs should be specific for the microbial targets, i.e., a high sequence specificity.

Based on knowledge about bacterial physiology the present inventors have focused the evaluation of potential gene targets on the following three major process complexes:

Cell wall synthesis, Protein synthesis (translation) , and Nucleic acid synthesis For each potential gene target within each process the following four points are addressed during evaluation:

The gene should be essential for bacterial survival.

The gene should occur as a single copy only.

The organism should not have physiological pathways that can compensate for the knock out of the target.

The target gene sequence should have no homology to the human genome.

A further consideration is that some physiological processes are primarily active in dividing cells whereas others are running under non-dividing circumstances as well. The present inventors have shown how to select targets from both groups.

1. Cell wall biosynthesis:

The target area for antibiotics in cell wall biosynthesis is the polymerization of the peptidoglycan layer, the so- called murein sacculus, which is a single layer in the Gram negative bacteria and multiple layers in the Gram positive. These targets are not present in cell wall-less bacteria {Mycoplasma spp. ) and hardly accessible in bacteria with impenetrable walls (Mycobacteria) . In some bacteria cell wall biosynthesis targets are inaccessible to some compounds, e.g. the glycopeptide vancomycin cannot penetrate the wall of Gram negative bacteria.

Interfering with cell wall biosynthesis targets is only effective in dividing cells. Additionally, the effect is dependent on the subsequent activation of murein- hydrolases leading to cell lysis. Furthermore, a low os olarity medium is required, because otherwise cell wall-less L-forms will be formed. The target proteins in cell wall biosynthesis are penicillin binding proteins, PBPs, the targets of, e.g., the beta-lactam antibiotic penicillin. They are involved in the final stages of cross-linking of the murein sacculus. By binding of penicillin, which acts- as a substrate analogue, PBP's are inhibited, and subsequently, hydrolytic enzymes are activated by the accumulation of peptidoglycan intermediates, thus hydrolysing the peptidoglycan layer and causing lysis.

E. coli has 12 PBPs, the high molecular weight- PBPs: PBPla, PBPlb, PBPlc, PBP2 and PBP3, and seven low molecular weight PBPs, PBP 4-7, DacD, AmpC and AmpH. Only the high molecular weight PBPs are known to be essential for growth and have therefore been chosen as targets for PNA antisense.

2. Protein synthesis (translation):

Targets in the area of protein synthesis are mainly found in the prokaryotic 70S ribosomes, i.e. either the 30S or the 50S subunit. Since protein biosynthesis is an important process throughout the bacterial growth cycle, the effect of hitting these targets is not dependent on cell division.

The selected targets, i.e. translation initiation, elongation and - release factors, are not known as targets for naturally occurring antibiotics.

3. Nucleic acid synthesis:

Both DNA and RNA synthesis are targets for antibiotics. A known target protein in DNA synthesis is gyrase. Gyrase is a topoisomerase which catalyzes negative supercoiling of the bacterial chromosome. When the enzyme is inhibited by antimicrobial agents such as quinolones, no supercoiling occurs and as a result of this the /27262

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chromosome cannot be packed into the new cell. Gyrase acts in replication, transcription, repair and restriction. The enzyme consists of two subunits, both of which are candidate targets for PNA.

In accordance with the above consideration, which are by no way limiting to the present invention, the present inventors have as an example selected the following potential targets.

Targets primarily activated in dividing cells:

rpoD Primary sigma factor, sigma 70 Transcπpuon (RNA polymerase subunit)

mrcA (ponA) Penicillin binding protein la Cell wall biosynthesis (PepUdoglycan sythetase) mrcB (ponB, pbpF) Penicillin binding protein lb Cell wall biosynthesis (Peptidoglycan sythetase)

mrdA Penicillin binding protein 2 Cell wall biosynthesis (Transpeptidase)

ftsl (pbpB) Penicillin binding protein 3 Cell wall biosynthesis

flsQ Cell division protein Cell division (ingrowth of wall at septum) ftsA ATP-bindmg cell division protein Cell division (septation process) ftsZ Cell division protein Cell division

(formation of circumferential ring) gyrA Gyrase, subunit A DNA replication, transcnption. (Topoisomerase II) repair

gyrB Gyrase, subunit B DNA replicaUon. transcription, repair 01/27262

1 6

Targets activated in non-dividing cells

infii EF1 Translation initiation factor inβ IF2 inf IF3 tufA/tuβ EF-Tu Translation elongation factor tsf EF-Ts

fusA EF-G p fA RF1 Translation release factor prβ RF2 prJ RF3

PNA's used for target gene selection.

The selection of target sequences include chemical considerations pertaining to the stability of the ensuing complexes between mRNA and PNA.

Initially for each target gene two overlapping sequences including the AUG start codon were synthetized. For optimal behaviour a balanced purine to pyrimidine ratio is necessary. Low purine content results in low affinity while high purine content often results in PNA aggregation.

For ease of bacterial uptake all PNA's were synthetized with a deca-peptide linked to the carboxy terminal. The overall structure of the peptide-PNA construct is KFFKFFKFFK-Ado-PNA-NH2 by way of example. Other peptides suitable for enhanced transport of the PNA over the cell wall may be used instead.

Table 1 depicts the peptide-PNA' s used for target gene selection experiments (start codons highlighted) .

Table 1

PNAs targeting essential genes in E. coli

Gene target PA number PNA sequence

sigma70 105 H-KFFKFFKFFK-Ado-TTG CTC CAT AAG-NH₂ 106 H-KFFKFFKFFK-Ado-CAT AAG ACG GTA-NH₂

PBPlal 103 H-KFFKFFKFFK-Ado-TAG TTT CAT TTG-NH₂ 104 H-KFFKFFKFFK-Ado-CAT TTG GGC AGT-NH₂

PBPla2 107 H-KFFKFFKFFK-Ado-GAA CTT CAC TGG-NH₂ 108 H-KFFKFFKFFK-Ado-CAC TGG AAA TTT-NH₂

PBPlblatgl 101 H-KFFKFFKFFK-Ado-CCC GGC CAT GCT-NH₂

102 H-KFFKFFKFFK-Ado-GGC CAT GCT TTT-NH₂

PBP2 95 H-KFFKFFKFFK-Ado-TAG TTT CAT CCG-NH₂

96 H-KFFKFFKFFK-Ado-TTT CAT CCG CTG-NH₂

PBP3 97 H-KFFKFFKFFK-Ado-TGC TTT CAT GCG-NH₂

ftsQ 113 H-KFFKFFKFFK-Ado-CTG CGA CAT ATT-NH₂

114 H-KFFKFFKFFK-Ado-CGA CAT ATT AGT-NH₂

ftsA 111 H-KFFKFFKFFK-Ado-CTT GAT CAT TGT-NH₂

112 H-KFFKFFKFFK-Ado-GAT CAT TGT TGT-NH₂

ftsZ 109 H-KFFKFFKFFK-Ado-TTC AAA CAT AGT-NH₂ 110 H-KFFKFFKFFK-Ado-ACA TAG TTT CTC-NH₂ 01/27262

gyrase A 133 H-KFFKFFKFFK-Ado-AAG GTC GCT CAT-NH₂ gyrase B 131 H-KFFKFFKFFK-Ado-AGA ATT CGA CAT-NH₂

IF1 129 H-KFFKFFKFFK-Ado-TGG_ CCA TCT AAT-NH₂

130 H-KFFKFFKFFK-Ado-CCA TCT AAT CCT-NH₂

IF2 127 H-KFFKFFKFFK-Ado-ACA TCT GTC ATG-NH₂

128 H-KFFKFFKFFK-Ado-TGT CAT GCT GTT-NH₂

IF2beta 125 H-KFFKFFKFFK-Ado-TTG ATT GCT CAC-NH₂

126 H-KFFKFFKFFK-Ado-TTG CTC ACT TTG-NH₂

IF2gamma 123 H-KFFKFFKFFK-Ado-TAG TCA TAT CGT-NH₂

124 H-KFFKFFKFFK-Ado-CAT ATC GTC TTG-NH₂

IF3 121 H-KFFKFFKFFK-Ado-CGC CTT TAA TAC-NH₂

122 H-KFFKFFKFFK-Ado-TAA TAC CTT ATT-NH₂

EF-Tu ( tufA) 119 H-KFFKFFKFFK-Ado-AGA CAC GGC TAT-NH₂

120 H-KFFKFFKFFK-Ado-CAC GGC TAT ATT-NH₂

EF-Tu ( tufB ) 117 H-KFFKFFKFFK-Ado-AGA CAT CGA TTG-NH₂ 118 H-KFFKFFKFFK-Ado-CAT CGA TTG TCC-NH₂

EF-Ts 115 H-KFFKFFKFFK-Ado-AGC CAT TCT AAA-NH₂ 116 H-KFFKFFKFFK-Ado-CAT TCT AAA ATC-NH₂

EF-G 147 H-KFFKFFKFFK-Ado-ACG AGC CAT TTG-NH₂

148 H-KFFKFFKFFK-Ado-AGC CAT TTG TTT-NH₂

RF- 1 150 H-KFFKFFKFFK-Ado-CAT AGG CGT AAA-NH₂

RF-2 151 H-KFFKFFKFFK-Ado-AAT TTC AAA CAT-NH₂

152 H-KFFKFFKFFK-Ado-CAT GGT CTG ATT-NH₂ 01/27262

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Rf-3 153 H-KFFKFFKFFK-Ado-AGA CAA CGT CAT-NH₂ 154 H-KFFKFFKFFK-Ado-AAC GTC ATA ATT-NH₂

Nonsense 136 H-KFFKFFKFFK-Ado-TGA CTA GAT GAG-NH₂

K is the one letter code for lysine (Lys) and F the one letter code for phenylalanine (Phe) . A, C, G and T denote the bases adenine, cytosine, guanine and thymine in the PNA sequence.

It is emphasised that the peptide KFFKFFKFFK is One of many possible peptides which may enhance the transport of the PNA over the cell wall. Other peptides are described in the co-pending application no. .

Other potential target genes are antibiotic resistance- genes. The skilled person would readily know from which genes to choose. Two examples are genes coding for beta- lactamases inactivating beta-lactam antibiotics, and genes encoding chloramphenicol acetyl transferase.

PNA's against such resistance genes could be used against resistant bacteria.

Infectious diseases are caused by microorganisms belonging to a very wide range of bacteria, viruses, protozoa, worms and arthropods and from a theoretical point of view PNA can be modified and used against all kinds of RNA in such microorganisms, sensitive or resistant to antibiotics.

Examples of micro-organisms which may be treated in accordance with the present invention are Gram-positive organisms such as Streptococcus, Staphylococcus, Peptococcus, Bacillus, Listeria, Clostridium, Propionebacteria, Gram-negative bacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella, Salmonella, Shigella, Proteus, Pseudomonas, Vibrio, Legionella, Haemophilus, Bordetella, Brucella, Campylobacter, Neisseria, Branhamella, and orgamisms which stain poorly or not at all with Gram's stain such as Mycobacteria, Treponema, Leptospira, Borrelia, Mycoplasma, Clamydia, Rickettsia and Coxiella,

The ability of PNAs to inhibit bacterial growth may be measures in many ways. The skilled person would readily know how to perform. As an example, the present inventors have chosen to measure the growth by the use of a microdilution broth method according to NCCLS guidelines.

An example could be:

Bacterial strain. E. coli K12 MG1655

Media: 10% Mueller-Hinton broth, diluted with sterile water.

Trays: 96 well trays, Costar # 3474, Biotech Line AS, Copenhagen. (Extra low sorbent trays are used in order to prevent / minimize adhesion of PNA to tray surface)

A logphase culture of E. coli is diluted with fresh prewarmed medium and adjusted to defined OD (here: Optical Density at 600 nm) in order to give a final concentration of 5xl0⁵ and 5xl0⁴ bacteria/ml medium in each well, containing 200 μl of bacterial culture. PNA is added to the bacterial culture in the wells in order to give final concentrations ranging from 300 nM to 1000 nM. However, for several of the initial syntheses we were faced with limited material and it was therefore necessary to select fewer than the ideal number of test concentrations. Trays are incubated at 37°C by shaking in a robot analyzer, PowerWave_x software KC' Kebo.Lab, Copenhagen, for 16 h and optical densities are measured at 600 nm during the incubation time in order to record growth curves. Wells containing bacterial culture without PNA are used as controls to ensure correct inoculum size and bacterial growth during the incubation. Cultures are tested in order to detect contaminations.

The individual peptide-PNA constructs have MW's between approx. 4200 and 5000 depending on composition. Therefore all tests were performed on a molar basis rather than a weight/volume basis. However, assuming an average- MW of the construct of 4500 a concentration of 500 nM equals 2.25 microgram/ml .

If different peptide-PNA construct were to be used, for example larger peptides and/or larger PNAs, the MW should be calculated and used accordingly.

Definition of growth inhibitory effect of PNA-constructs : The bacterial growth in the wells is described by the lagphase i.e. the period until (before) growth starts, the logphase i.e. the period with maximal growth rate, the steady-statephase followed by the deathphase. These parameters are used when evaluating the inhibitory effect of the PNA on the bacterial growth, by comparing growth curves with and without PNA. The intra- and interassay coefficient of variation on the OD measurements were 4,5% and 8%, respectively.

Total inhibition of bacterial growth may be defined as: OD (16h) = OD (Oh) +/- 8%.

The peptide is normally linked to the PNA sequence via the amino or carboxy end. However, the PNA sequence may also be linked to an internal part of the peptide.

Preferable, the PNA sequence may be linked to the C- terminal end of the peptide.

The PNA molecule may be connected to the Peptide moiety through a direct binding or through a linker. A variety of linking groups can be used to connect the PNA with the Peptide. The choice of linking groups is not important for the present invention. However, some linking groups may be advantageous in connection with specific combinations of PNA and Peptide. The skilled person would readily be able to choose the right linkers. Some linking groups are described in WO 96/11205 and W098/52614, the content of which are hereby incorporated by reference.

Examples of linking groups are Ado (8-amino-3, 6- dioxaoctanoic acid), c cc (cystein-4- (N- maleimidomethyl) cyclohexane-1-carboxylic acid), ahex (6- aminohexanoic acid) , 4-aminobutyric acid, 4- a inocyclohexylcarboxylic acid, polyethylene glycols and amino acids. Any of these groups may be used as a single linking group or together with more groups in creating a suitable linker arm. Further, the different linking groups may be combining in any order and number in order to obtain different functionalities in the linker arm.

The modified PΝA molecule according to the present invention comprises a PΝA oligomer of a sequence which is complementary to at least one target nucleotide sequence in a micro-organism, such as a bacteria. The target may be a nucleotide sequence of any RΝA which is essential for the growth and/or reproduction of the bacteria. Alternatively, the target may be a gene encoding a factor responsible for resistance to antibiotics. In a preferred embodiment, the functioning of the target nucleotide sequence is essential for the survival of the bacteria and the functioning of the target nucleic acid is blocked by the PΝA sequence, in an antisense manner. The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations, anti-parallel or parallel. As used in the present invention, the term complementary as applied to PNA does not in itself specify the orientation parallel or anti-parallel. It is significant that the most stable orientation of PNA/DNA and PNA/RNA is anti-parallel. In a preferred embodiment, PNA targeted to single strand RNA is complementary in an anti-parallel orientation.

In a another preferred embodiment of the invention a bis- PNA consisting of two PNA oligomers covalently linked to each other is targeted to a homopurine sequence (consisting of only adenine and/or guanine nucleotides) in RNA (or DNA) , with which it can form a PNA₂-RNA (PNA₂- DNA) triple helix.

In a preferred embodiment of the invention, the PNA contains from 5 to 20 nucleobases, in particular from 7- 15 nucleobases, and most particular from 9 to 12 nucleobases .

Peptide Nucleic Acids are described in WO 92/20702 and WO 92/20703, the content of which are hereby incorporated by reference.

The modified PNA molecules may initially be screening in the sensitive 10% medium assay. Positive results are then run in the 100% medium assay in order to verify the inhibitory effect in a more "real" environment (cf the American guidelines (NCCLS) ) .

After the initially screening procedures, it may be advantageous to look for even better targets that the ones initially identified. This optimisation from a hit may take many forms. One way may be to look for better targets in the gene identified as a potentially target. This can be done by a random trial and error method or by a more systematic genewalk. The advantage in using a genewalk is that almost all possibilities are tried out. On the other hand a lot of constructs are needed in order to cover the gene.

In accordance with the present invention, the modified PNA molecules can be used to identify preferred targets for the PNA. Based upon the known or partly known -genome of the target micro-organisms, e.g. from genome sequencing or cDNA libraries, different PNA sequences can be constructed and linked to an effective anti-infective enhancing Peptide and thereafter tested for its anti- infective activity. It may be advantageous to select PNA sequences shared by as many micro-organisms as possible or shared by a distinct subset of micro-organisms, such as for example Gram-negative or Gram-positive bacteria, or shared by selected distinct micro-organisms or specific for a single micro-organism.

In a further aspect of the present invention, the invention provides the possibility of selecting gene targets which may be used in creating new anti-infective drugs, such as bacteriostatics, in form of antisense PNA sequences conjugated to activity enhancing moieties, for example transport enhancing peptides. Such conjugates may be formulated in compositions for use in inhibiting growth or reproduction of infectious micro-organisms. In one embodiment, the inhibition of the growth of microorganisms is obtained through treatment with either the modified PNA molecule alone or in combination with antibiotics or other anti-infective agents. In another embodiment, the composition comprises two or more different modified PNA molecules. A second modified PNA molecule can be used to target the same bacteria as the first modified PNA molecule or in order to target different bacteria. In the latter form, specific combinations of target bacteria may be selected to the treatment. Alternatively, the target can be one or more genes which confer resistance to one or more antibiotics to one or more bacteria. In such a treatment, the composition or the treatment further comprises the use of said antibiotic (s) .

The compositions may include pharmaceutically acceptable carriers and/or diluents. Such carrier and diluents are known in the art. The active compositions may be administered in form of tablets, injections, powders, solutions, sprays, dressings, etc.

In formulations for treatment or prevention of infectious diseases in mammals the amount of active modified PNA molecules used is determined in accordance with the specific active drug, organism to be treated and carrier of the organism.

In yet a further aspect of the present invention, the present invention concerns the provision of modified PNA molecules for use in disinfecting objects other than living beings, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools and the like.

The following examples are merely illustrative of the present invention and should not be considered limiting of the scope of the invention in any way. The principle of the present invention is shown using E. coli as a test organism. However, as shown in Example 18, the advantageous effect applies in the same way to other bacteria.

Example 1

Description of a primary screen

The bacterial growth assay is designed to identify PNAs that inhibit or completely abolish bacterial growth. Growth inhibition results from antisense binding of PNA to mRNA of the targeted gene. The test compound (PNA) is present during the whole assay.

Components

The experimental bacterial strain for the protocol is

Escherichia coli K12 MG1655 (E. coli Genentic Stock Center, Yale University, New Haven) . The medium for growth is 10% sterile LB (Lurea Bertani) medium. E. coli test cells are pre-cultured in LB medium at 37 °C over night (over night culture) . The screen is performed in 96-well microtiter plates at 37 °C under constant shaking.

PNAs are dissolved in H₂0 as a 40x concentrated stock solution.

Assay conditions From an over night culture a fresh culture (test culture) is grown to mid-log-phase (OD₆oo = 0.1 corresponding to 10⁷ cells/ml) at 37 °C. The test culture is diluted stepwise in the range 10⁵ to 10¹ with 10% LB medium. 195 ul of diluted cultures plus 5 ul of a 40x concentrated PNA stock solution are added to each test well.

96-well microtiter plates are incubated in a microplate scanning spectrophotometer at 37 °C under constant shaking. OD₆oo measurements are performed automatically every 3.19 minutes and recorded simultaneously.

Target genes: Penicillin binding proteins (PBPs)

PBPs act in biosynthesis of murein (peptidoglycan) , which is part of the envelope of Gram-positive and Gram- negative bacteria. By binding of penicillin, which acts as substrate analogon, PBP' s are inhibited, and subsequently, hydrolytic enzymes are activated by the accumulation of peptidoglycan intermediates, thus hydrolysing the peptidoglycan layer and causing lysis.

PNA design no. 1

PNA #PNA26 has been designed according to the sequence of the mrcA (ponA) gene of E. coli , encoding PBP1A. The sequence of the mrcA gene (accession number X02164) was obtained from the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al. 1985, Eur J Biochem 147:437-46). The sequence of the mrcA gene is shown in Figure 3.

The target region of #PNA26 is the following:

sense 5' AATGGGAAATTTCCAGTGAAGTTCGTAAAG 3' 121 + + + 150 antisense 3' TTACCCTTTAAAGGTCACTTCAAGCATTTC 5'

Both the coding and the non-coding (antisense) strand of the GTG start codon region are shown.

The sequence of the GTG start codon region of the antisense strand and PNA26 are shown in the 5' to 3' orientation:

antisense 5' CTTTACGAACTTCACTGGAAATTTCCCATT 3' PNA26 H-KFFKFFKFFK-Ado-CACTGGAAATTT-Lys-NH₂

PNA26 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide. Growth assay with PNA26

The assay was performed as follows:

Dilutions of the test culture corresponding to 10⁵, 10⁴, 10³, 10² and 10¹ cells/ml containing PNA26 at a final concentration of 1.5, 2.0, 2.5, 3.0 and 3.5 uM are incubated at 37°C for 16 hours with constant shaking.

Total inhibition of growth can be seen in cultures with lC'-lO¹ cells/ml and a PNA concentration of at least 2.5uM (Table 2) .

PNA design no. 2

PNA #PNA14 has been designed according to the sequence of the mrdA gene _. encoding PBP2. The sequence (accession number AE000168, bases 4051-5952) was obtained from the E. coli genome database at the NCBI (Genbank, National Centre for Biotechnology Information, USA) . The sequence of the mrdA gene is shown in Figure 4.

The target region of PNA14 is the following:

sense 5' GAGTAGAAAACGCAGCGGATGAAACTACAGAAC 3'

antisense 3' CTCATCTTTTGCGTCGCCTACTTTGATGTCTTG 5'

Both the coding (sense) and the non-coding (antisense) strand of the GTG start codon region are shown.

In the following the sequence of the ATG start codon region of the antisense strand and PNA26 are shown in the 5' to 3' orientation:

antisense 5' GTTCTGTAGTTTCATCCGCTGCGTTTTCTACTC 3' PNA14 H-KFFKFFKFFK-Ado-TTTCATCCGCTG-Lys-NH? PNA14 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid peptide.

Growth assay with PNA14

The assay was performed as follows:

Dilutions of the test culture corresponding to 10⁵, 10^"1, 10³, 10² and 10¹ cells/ml containing PNA14 at a final concentration of 1.3, 1.4 and 1.5 uM are incubated at 37°C for 16 hours with constant shaking. Total inhibition of growth can be seen in cultures with IC'-IO¹ cells/ml and a PNA concentration of at least 1.4uM (Table 3) .

Example 2

A number of constructs taken from Table 1 were tested in accordance with the experimental setup and discussed on page 20. As exemplified in Figures 5 and 6 the bacterial growth conditions chosen for the testing of PNA- constructs allowed a direct comparison of the inhibitory effect exhibited by different PNA specificities. Both figures show bacterial growth curves obtained without PNA-peptide and with PNA-peptide present at final concentrations varying from 200 nM to 1000 nM.

The PNA #109 in Figure 5 is directed against the ftsZ gene encoding a cell division protein. The PNA #111 in Figure 6 is directed at the ftsA gene encoding an ATP- binding protein involved in the septation process during bacterial cell division. The inhibitory effect is dose dependent for both constructs. Complete inhibition of the bacterial growth was observed with 600 nM for PNA #109 and with 1000 nM for PNA #111 (1.5 x 10⁵ bacteria/ml) .

The Tables 4 and 5 below are summaries of the bacterial growth inhibition assays performed with the aim of

Table 2 Bacterial growth inhibition with PNA 26; E. coli K12 in 10% Mueller-Hinton broth.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times nd: Not done o

PNA PNA cone, in wells nM

1300 1400 1500

Bacterial 1% 0.1% 0.1% 0.001% 1% 0.1% 0.1% 0.001% 1% 0.1% 0.1% 0.001% concentration 0.0001% 0.0001% 0.0001%

14 - - - - (+) + + + + + + +

+ +

Table 3 Bacterial growth inhibition with PNA 14; E. coli K12 in 10% Mueller-Hinton broth.

+ : Total inhibition of bacterial growth

(+): Significantly extended lagphase, (more than five times)

Lagphase extended less than five times, nd: Not done

Table 4 Growth inhibitory effect of PNA's against target genes in E. coli K12.

+ : Total inhibition of bacterial growth (+ ) : Significantly extended lagphase, (more than five times!

Lagphase extended less than five times nd: Not done

Table 5 Growth inhibitory effect of PNA's against target genes in E. coli Kl

+ : Total inhibition of bacterial growth

(+) : Significantly extended lagphase, (more than five times)

- : Lagphase extended less than five times nd: Not done

selecting target genes.

As outlined in Tables 4 and 5 above, PNA-peptide constructs against several of the selected potential target genes were able to inhibit bacterial growth within the concentration range chosen.

The "nonsense" PNA i.e. # 136 did not fully match any genomic region in the organism and hence did not allow a full length base pairing. This control was used to investigate general toxicity of peptide-PNA constructs. No bacterial growth inhibition was detected within the concentration range chosen.

Targets involved in bacterial cell division:

At a concentration of 1000 nM several of the PNA- constructs against the potential targets exhibited bacterial growth inhibition. Some of these behaved in a similar fashion e.g. Gyrase A, Gyrase B, PBP2 and PBP3. Comparing PBPla2 and ftsA the latter showed effect on growth with two different PNA's where the first was effected by only one PNA. ftsA was a stronger target than Sigma 70. ftsA as well as ftsZ were stronger targets than PBPlblatgl. A direct comparison between ftsA and ftsZ indicated that they were both suitable targets, however, the inhibitory effect seen with PNA-constructs against ftsZ were stronger than for ftsA (see Table 5 and figures 4 and 5) . In this way a primary target, f sZ, which encodes a cell division protein, has been selected.

Targets active in non-dividing bacterial cells:

At concentrations down to 500 nM PNA-constructs against several of the potential targets i.e. IF1, IF3, EF-G and F2 exhibited bacterial growth inhibition. However, at 500 nM none of the PNA's inhibited the bacterial growth completely. At a concentration of 700 nM PNA's against three targets i.e. IF1, IF 2 beta and Ef-Ts exhibited a complete inhibition of bacterial growth. Among targets tested with PNA's at 1000 nM, one (RF2) was inhibited by just one PNA, and two (IF1 and EF-G) were inhibited by both PNA's tested. IF1 and EF-G would both be suitable gene targets as judged from the bacterial growth inhibition experiments. However, bearing the subsequent genewalk in mind, we have chosen the short gene {infA encoding IF1, 230 bases) over the longer gene" { fusA encoding EF-G, 2 kilobases) as our primary target.

Example 3

Genewalk on the gene encoding IF-1 of E. coli ,

The selected target genes may be further analyzed by genewalk to select the optimal target sequence within each gene. The PNA^'s directed at these sequences may be in experiments aimed at designing a suitable bacterial uptake enhancing compound.

Because of the good bacterial growth inhibition with the PNA # 130 against the gene encoding IF-1 of E. coli , a manual genewalk was made. Table 6 shows the different PNA sequences designed from the infA gene. The experimental setup was as described above comprising the use of E. coli K12 MG1655 in 10% Mueller-Hinton broth.

235 H-KFFKFFKFFK-Ado-AGT CAC TTT GTC-NH₂

236 H-KFFKFFKFFK-Ado-AAC CAC GTG ACC-NH₂

237 H-KFFKFFKFFK-Ado-GAA CAT GGT ATT-NH₂

238 H-KFFKFFKFFK-Ado-TTG CAT TTC AAT-NH₂

239 H-KFFKFFKFFK-Ado-CTA ATC CTC TGG-NH₂

240 H-KFFKFFKFFK-Ado-CAT CTA ATC CTC-NH₂

241 H-KFFKFFKFFK-Ado-GCC ATC TAA TCC-NH₂

242 H-KFFKFFKFFK-Ado-TCT AAT CCT CTG-NH₂

243 H-KFFKFFKFFK-Ado-ATC TAA TCC TCT-NH₂

244 H-KFFKFFKFFK-Ado-GTA TCA CTA CCG-NH₂

245 H-KFFKFFKFFK-Ado-GCT CAG GTC GTA-NH₂

246 H-KFFKFFKFFK-Ado-AGC GAC TAC GGA-NH₂

264 H-KFFKFFKFFK-Ado-GGC CAT CTA ATC-NH2

265 H-KFFKFFKFFK-Ado-GAC AAT GCG GCC-NH2

266 H-KFFKFFKFFK-Ado-GGT CAG TTC AAC-NH2

267 H-KFFKFFKFFK-Ado-CCG TCA GGA TGC-NH2

268 H-KFFKFFKFFK-Ado-GCG GAT GTA GTT-NH2

269 H-KFFKFFKFFK-Ado-TTA CCG GAG ATG-NH2

270 H-KFFKFFKFFK-Ado-TTC TAA CTC TAC-NH2

271 H-KFFKFFKFFK-Ado-GGC AAC GTT TCA-NH2

272 H-KFFKFFKFFK-Ado-ACC GGC AAG ATA-NH2

273 H-KFFKFFKFFK-Ado-AAC GGT ACC TTG-NH2

274 H-KFFKFFKFFK-Ado-ATT GTC TC TTT-NH2

275 H-KFFKFFKFFK-Ado-GTG CAG TAA CCA-NH2

The results are shown in Table 7, indicating that the construct PNA 267 would be the potentially best compound for use in targeting the E. coli gene infA.

ON

TABLE 7 + : Total inhibition of bacterial growth

(+) : Significantly extended lagphase, (more than five times) - : Lagphase extended less than five times nd: Not done

References

Davies, J. et al . (1994) Science 264, 375-82.

Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O. Science (1991) 254, 1457-1500.

Egholm, M, Buchardt, 0, Christensen, L, Behrens, C, Freier, S. M. Driver, D.A., Berg, R.H., Kim, S.K.,

Norden, B. and Nielsen, P.E. Nature (1993) 365, 566-568.

Demidov, V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, 0. Sonnichsen, H. S. and Nielsen, P.E. Biochem. Pharmacol. (1994) _4_8, 1310-1313.

Nielsen, P.E. and Haaima, G. Chemical Society Reviews (1997) 73-78.

Hanvey et al. Science (1992) 258, 1481-5.

Knudsen, H. and Nielsen, P.E. Nucleic Acids Res. (1996) 24, 494-500.

Lewis, L.G. et al . Proc. Natl. Acad. Sci. USA (1996) 93, 3176-81.

Meyer, O. et al. J. Biol. Chem. (1998) 273, 15621-7.

Nyce, J.W. and Metzger, W.J. Nature (1997) 385 721-725.

Pooga, M. et al, Nature Biotechnology (1998) 16, 857-61.

Good, L. & Nielsen, P.E. Proc. Natl. Acad. Sci. USA (1998) 95, 2073-2076. Good, L. & Nielsen, P.E. Nature Biotechnology (1998) 16, 355-358.

Broome-Smith et al . 1985, Eur J Biochem 147:437-46.

Egholm,M.; Dueholm,K. L.; Buchardt, 0.;Coull, J.; Nielsen, P. E . ; Nucleic Acids Research 1995, 23,217-222.

Claims

1. A method of identifying one or more target genes in a micro-organism, which target gene(s) may be the basis for an anti-infective treatment, comprising:

a) selecting potential target genes known to be present in the micro-organism,

2. A method according to claim 1, wherein the PNA sequence comprises an activity enhancing moiety.

3. A method according to claim 2, wherein the activity enhancing moiety is a peptide.

4. A method according to claim 2 or claim 3, wherein the activity enhancing moiety is a moiety which enhances the access of the PNA to the gene target in the micro- organism.

5. A method according to claim 4, wherein the activity enhancing moiety enhances the crossing of the PNA over the cell wall of the micro-organism.

6. A method according to any of claims 3-5, wherein the peptide is (Lys-Phe-Phe) ₃-Lys .

7. Use of a target gene or a target gene product as the basis for a preparation of an anti-infective agent, said target gene being identifiable by a method according to any of claims 1-6.

8. Use according to claim 7, wherein the anti-infective agent is a nucleotide sequence complementary to at least a part of the target gene.

9. Use according to claim 8, wherein the nucleotide sequence is an antisense PNA.

10. Use according to claim 9, wherein the PNA further comprises an activity enhancing moiety.

11. Use according to claim 10, wherein the activity enhancing moiety is a peptide.

12. Use according to claim 10 or claim 11, wherein the activity enhancing moiety is a moiety which enhances the access of the PNA to the gene target in the microorganism.

13. Use according to claim 12, wherein the activity enhancing moiety enhances the crossing of the PNA over the cell wall of the micro-organism.

14. Use according to any of claims 11-13, wherein the peptide is (Lys-Phe-Phe) ₃-Lys .

15. Use according to any of claims 9-14, wherein the resulting antisense PNA is not complementary to any human or animal DNA sequence.

16. Use according to any of claims 7-15, wherein the target gene is a single copy gene.

17. Use according to any of claims 9-16, wherein the antisense PNA is not complementary to more than one target site.

18. Use according to any of claims 7-17, wherein the micro-organism is E. coli.

19. Use according to claim 18, wherein the target gene is selected from the group consisting of ftsA, infA, infB, infC, tsf, fusA and prfB.

20. A PNA molecule comprising a PNA sequence which is complementary (antisense) to at least a part of a target gene in a micro-organism, said target gene being identifiable by a method according to any of claims 1-6.

21. A PNA molecule according to claim 20, wherein the PNA further comprises an activity enhancing moiety.

22. A PNA molecule according to claim 21, wherein the activity enhancing moiety is a peptide.

23. A PNA molecule according to claim 21 or claim 22, wherein activity enhancing moiety is a moiety which enhances the access of the PNA to the gene target in the micro-organism.

24. A PNA molecule according to claim 23, wherein the activity enhancing moiety enhances the crossing of the PNA over the cell wall of the micro-organism.

25. A PNA molecule according to any of claims 22-24, wherein the peptide is (Lys-Phe-Phe) ₃-Lys .

26. A PNA molecule according to any of claims 20-25, wherein the PNA is not complementary to any human or animal DNA sequences.

27. A PNA molecule according to any of claims- 20-26, wherein the target gene is a single copy gene.

28. A PNA molecule according to any of claims 20-27, wherein the antisense PNA is not complementary to more than one target site.

29. A PNA molecule according to any of the claims 20-28, wherein the micro-organism is E.coli.

30. A PNA molecule according to claim 29, wherein the target gene is selected from the group consisting of ftsA, infA, infB, infC, tsf, fusA and prfB.

31. A PNA molecule according to claim 30, wherein the PNA sequence is selected from:

TAGTTTCATTTG, CATTTGGGCAGT, GAACTTCACTGG, CACTGGAAATTT,

CCCGGCCATGCT, GGCCATGCTTTT, TAGTTTCATCCG, TTTCATCCGCTG, TGCTTTCATGCG, CTGCGACATATT, CGACATATTAGT, CTTGATCATTGT,

GATCATTGTTGT, TTCAAACATAGT, ACATAGTTTCTC, AAGGTCGCTCAT,

AGAATTCGACAT, TGGCCATCTAAT, CCATCTAATCCT, ACATCTGTCATG,

TGTCATGCTGTT, TTGATTGCTCAC, TTGCTCACTTTG, CATATCGTCTTG,

CGCCTTTAATAC, AGACACGGCTAT, CACGGCTATATT, AGACATCGATTG, CATCGATTGTCC, AGCCATTCTAAA, CATTCTAAAATC, ACGAGCCATTTG,

AGCCATTTGTTT, CATAGGCGTAAA, AATTTCAAACAT, CATGGTCTGATT, AGACAACGTCAT and AACGTCATAATT .

32. A PNA molecule according to claim 31, wherein the PNA sequence is selected from:

CTTGATCATTGT, TTCAAACATAGT, CCATCTAATCCT, AGCCATTTGTTT and CATGGTCTGATT

33. A PNA molecule selected from the group consisting of:

H-KFFKFFKFFK-Ado-TAG TTT CAT TTG-NH₂, H-KFFKFFKFFK-Ado-CAT TTG GGC AGT-NH₂, H-KFFKFFKFFK-Ado-GAA CTT CAC TGG-NH₂, H-KFFKFFKFFK-Ado-CAC TGG AAA TTT-NH₂, H-KFFKFFKFFK-Ado-CCC GGC CAT GCT-NH₂, H-KFFKFFKFFK-Ado-GGC CAT GCT TTT-NH₂, H-KFFKFFKFFK-Ado-TAG TTT CAT CCG-NH₂, H-KFFKFFKFFK-Ado-TTT CAT CCG CTG-NH₂, H-KFFKFFKFFK-Ado-TGC TTT CAT GCG-NH₂, H-KFFKFFKFFK-Ado-CTG CGA CAT ATT-NH₂, H-KFFKFFKFFK-Ado-CGA CAT ATT AGT-NH₂, H-KFFKFFKFFK-Ado-CTT GAT CAT TGT-NH₂, H-KFFKFFKFFK-Ado-GAT CAT TGT TGT-NH₂, H-KFFKFFKFFK-Ado-TTC AAA CAT AGT-NH₂, H-KFFKFFKFFK-Ado-ACA TAG TTT CTC-NH₂, H-KFFKFFKFFK-Ado-AAG GTC GCT CAT-NH₂, H-KFFKFFKFFK-Ado-AGA ATT CGA CAT-NH₂, H-KFFKFFKFFK-Ado-TGG CCA TCT AAT-NH₂, H-KFFKFFKFFK-Ado-CCA TCT AAT CCT-NH₂, H-KFFKFFKFFK-Ado-ACA TCT GTC ATG-NH₂, H-KFFKFFKFFK-Ado-TGT CAT GCT GTT-NH₂, H-KFFKFFKFFK-Ado-TTG ATT GCT CAC-NH₂, H-KFFKFFKFFK-Ado-TTG CTC ACT TTG-NH₂, H-KFFKFFKFFK-Ado-TAG TCA TAT CGT-NH₂, H-KFFKFFKFFK-Ado-CAT ATC GTC TTG-NH₂, H-KFFKFFKFFK-Ado-CGC CTT TAA TAC-NH₂, H-KFFKFFKFFK-Ado-TAA TAC CTT ATT-NH₂,

H-KFFKFFKFFK-Ado-AGA CAC GGC TAT-NH₂,

H-KFFKFFKFFK-Ado-CAC GGC TAT ATT-NH₂,

H-KFFKFFKFFK-Ado-AGA CAT CGA TTG-NH₂, H-KFFKFFKFFK-Ado-CAT CGA TTG TCC-NH₂,

H-KFFKFFKFFK-Ado-AGC CAT TCT AAA-NH₂,

H-KFFKFFKFFK-Ado-CAT TCT AAA ATC-NH₂,

H-KFFKFFKFFK-Ado-ACG AGC CAT TTG-NH₂,

H-KFFKFFKFFK-Ado-AGC CAT TTG TTT-NH₂, H-KFFKFFKFFK-Ado-CAT AGG CGT AAA-NH₂,

H-KFFKFFKFFK-Ado-AAT TTC AAA CAT-NH₂,

H-KFFKFFKFFK-Ado-CAT GGT CTG ATT-NH₂,

H-KFFKFFKFFK-Ado-AGA CAA CGT CAT-NH₂ and

H-KFFKFFKFFK-Ado-AAC GTC ATA ATT-NH₂

34. A PNA molecule according to claim 33 selected from:

H-KFFKFFKFFK-Ado-CTT GAT CAT TGT-NH₂,

H-KFFKFFKFFK-Ado-TTC AAA CAT AGT-NH₂, H-KFFKFFKFFK-Ado-CCA TCT AAT CCT-NH₂,

H-KFFKFFKFFK-Ado-AGC CAT TTG TTT-NH₂ and H-KFFKFFKFFK-Ado-CAT GGT CTG ATT-NH₂

35. A gene segment identifiable according to the method of any of claims 1-6.

36. A gene segment according to claim 35 which may hybridize under stringent conditions to a PNA sequence according to claim 31.

37. Use of a gene target identifiable according to the method of any of claims 1-6 in the identification of compounds which may inhibit the growth of microorganisms .