WO2001044456A2 - Modification de micro-organismes - Google Patents

Modification de micro-organismes Download PDF

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Publication number
WO2001044456A2
WO2001044456A2 PCT/GB2000/004795 GB0004795W WO0144456A2 WO 2001044456 A2 WO2001044456 A2 WO 2001044456A2 GB 0004795 W GB0004795 W GB 0004795W WO 0144456 A2 WO0144456 A2 WO 0144456A2
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micro
organism
gene
vector
target
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PCT/GB2000/004795
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WO2001044456A3 (fr
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Catherine Elizabeth Dunn Rees
Philip John Hill
Christine Elizabeth Ruth Dodd
Laurence Stephen Tiley
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The University Of Nottingham
Cambridge University Technical Services Limited
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Priority to AU21966/01A priority Critical patent/AU2196601A/en
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Publication of WO2001044456A3 publication Critical patent/WO2001044456A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/126—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes involving RNAse P

Definitions

  • the present invention relates to the modification of micro-organisms and in particular but not exclusively to the modification of undesirable microorganisms using antisense bacteriophage.
  • Micro-organisms including bacteria and fungi, can present serious and unwanted problems in a wide range of industrial and natural environments. For instance they can be the cause of problems in the medical, dairy, food, paint, pharmaceutical and oil industries to name but a few.
  • antisense is used in this specification to include sequences that function in accordance with the methodologies as antisense sequences.
  • a method of modifying micro-organisms comprising introducing to a target micro-organism a vector comprising an antisense form of the whole or part of a target gene normally found in that micro-organism whereby upon infection of the micro-organism by the vector and expression of the antisense form of that gene or part gene in the host translation of the target gene is prevented or inhibited and the properties of the micro-organism thereby modified.
  • the method is preferably used to modify one or more undesirable properties or characteristics of a target micro-organism and may control the expression of one or more proteins normally involved in or the cause of the undesirable properties or characteristics.
  • the sequences targeted may encode proteins, involved for example in cell growth, cell resistance to chemicals such as antibiotics and/or viruler ce of a micro-organism allowing it to invade tissues of other organisms.
  • the method is used to prevent expression of a target gene to a significant degree in the micro-organism.
  • expression of the target gene is not significant to the micro-organism in the environment where control is not desired which is desirably its normal environment, but is significant in the environment in which the micro-organism is to be controlled.
  • the antisense form is introduced into the micro-organism in an environment in which the target gene is not expressed to any significant degree and remains in the micro-organism when it enters an environment in which the target gene is expressed to a significant degree.
  • the antisense form is desirably introduced in an environment in which the micro-organism does not or is unlikely to have the undesirable affect which is to be controlled.
  • the antisense form may be introduced to a micro-organism in an environment in which the target gene is significantly expressed, which environment is desirably that in which the micro-organism can have the undesirable affect which is to be controlled.
  • the method may comprise the use of a vector having antisense forms of a plurality of specific target genes and/or part genes which target genes may be normally found in a particular micro-organism such that in use the method modifies the properties of only that particular micro-organism.
  • the method may comprise the use of a vector having antisense forms of a plurality of target genes and/or part genes which target genes may be normally found in a plurality of respective micro-organisms, whereby the method can be used to modify the properties of one or more different micro-organisms.
  • the vector used may comprise a bacteriophage, which is preferably engineered to comprise the said antisense form(s).
  • the bacteriophage used may comprise a constitutive promoter to provide for constitutive expression of the antisense form(s) m the host micro-organism or an mducible promoter that is active when expression of the antisense form is desired.
  • the bacteriophage used is preferably constructed such that the antisense form(s) is/are expressed in the lysogemc state.
  • the vector In its lysogemc state the vector is preferably not detrimental to the micro-organism in its normal environment, and only has controlling effect when the infected micro-organism enters an environment in which the microorganism could express a property or characteristic to be controlled.
  • a vector for use in the modification of the properties or characteristics of a target microorganism comprising an antisense form of the whole or part of a specific target gene normally found in that micro-organism whereby upon infection of the micro-organism by the vector and expression of the antisense form of that gene or part gene in the micro-organisms translation of that target gene is prev ented or inhibited and the properties of the micro-organism thereby modified.
  • the vector used is a bacteriophage, which bacteriophage is genetically engineered to comprise the said antisense gene or part gene form of a specific target gene normally found in the target micro-organism.
  • the bacteriophage may comprise antisense gene or part gene forms of a plurality of specific target genes, which genes are normally found in a particular micro-organism.
  • the bacteriophage may comprise antisense gene or part gene forms of a plurality of target genes normally found in a plurality of different respective micro organisms
  • the ⁇ ector may be substantially as hereinbefore described.
  • a method of modifying the properties of micro-organisms comprising introducing to a target micro-organism a vector comprising an antisense gene or part gene form of a target gene normally found in that micro-organism whereby upon infection of the micro-organism by the vector the RNA transcribed from the antisense form targets the RNA of the target gene for degradation thereby preventing protein formation from the gene, the properties of the micro-organism thereby being modified.
  • the RNA transcribed from the antisense form targets the RNA. for degradation pathways m the micro-organism.
  • the degradation pathways may include cleavage of the target RNA by ⁇ bonucleases and/or nbozymes.
  • the RNA transcribed from the antisense form may form a ⁇ bozyme which will contain sequences specific to the target mRNA.
  • the ⁇ bozyme may be hairpm, hammerhead or others.
  • the antisense form may code for an external guide sequence (EGS) which may comprise a short RNA sequence which has in the order of eleven or more bases able to hybridise with the RNA of the target gene followed by CCA sequence at the 3' end.
  • EGS external guide sequence
  • hybridisation of the EGS to the target RNA produces a duplex molecule that mimics the substrate of Ribonuclease P and results in the cleav age of the target molecule.
  • the EGS or ⁇ bozyme targets the RNA for degradation by specific hybrid formation.
  • the method is preferably used to modify one or more undesirable properties or characteristics of a target micro-organism and may control the production of one or more proteins normally involved in the undesirable properties or characteristics.
  • the proteins targeted may be involved for example in cell growth, cell resistance to chemicals such as antibiotics and/or virulence of a micro-organism allowing it to mvade tissues of other organisms whereby to cause undesirable effects such as disease.
  • the method is used to block protein production from a target RNA before the RNA is translated to a significant degree m the micro-organism.
  • the target RNA is not produced in any significant amount or encodes a product that is not beneficial to the micro-organism until the micro-organism enters or experiences an environment in which the micro-organism would exhibit the undesirable properties or characteristics to be controlled.
  • the antisense form may be introduced to the micro-organism in an environment in which the micro-organism can have the undesirable affect to be controlled.
  • the method may comprise the use of a vector having antisense gene or part gene forms of a plurality of specific target genes which target genes may be normally found in a particular micro-organism such that in use the method modifies the properties of only that particular micro-organism.
  • the method may comprise use of a vector having antisense gene or part gene forms of a plurality of target genes which target genes may be normally found in a plurality of respective micro-organisms whereby the method can be used to modify the properties of one or more different microorganisms.
  • the vector used may comprise a bacteriophage, which is preferably engineered to comprise the said antisense form(s).
  • the vector may comprise features substantially as described above.
  • a vector for use in the modification of the properties of a micro-organism comprising an antisense form of the whole or part of a target gene normally found in that micro-organism, whereby upon infection of the micro-organism by the vector RNA transcribed from the antisense form targets the RNA of the target gene for degradation thereby preventing protein formation from the gene, the properties of the micro-organism thereby being modified.
  • the vector is substantially as described above, and may be genetically engineered bacteriophage which comprises the said antisense gene or part gene form of a specific target gene normally found in the target micro- organism.
  • the bacteriophage may comprise antisense gene or part gene forms of a plurality of specific target genes, which genes are normally found in a particular micro-organism.
  • the bacteriophage may comprise antisense gene or part gene forms of a plurality of target genes normally found in a plurality of different respective micro-organisms.
  • the bacteriophage In its lysogenic state the bacteriophage is preferably not detrimental to the micro-organism in its normal environment.
  • an antisense form may be coupled to a CCA or similar sequence at the 3' end thereof, and may comprise in the order of eleven or more bases.
  • the antisense form(s) is/are expressed in a lysogenic state of the bacteriophage.
  • a DNA construct for use in modifying the activity of a micro-organism comprising an antisense gene or part gene form of a specific target gene normally found in a target micro-organism.
  • the construct may comprise antisense gene or part gene forms of a plurality of specific target genes normally found in a particular micro-organism, or in a plurality of different respective micro-organisms.
  • the target genes are not expressed in the micro-organism to a significant degree.
  • expression of the target gene is not significant to the micro-organism in the environment where control is not desired which is desirably its normal environment, but is significant in the environment in which the micro-organism is to be controlled.
  • the target gene is not essential to the micro-organism in its normal environment.
  • a method of modifying the properties of target micro-organisms and a vector and DNA construct for use in such a method comprises introducing to a target micro-organism to be modified, a vector comprising an antisense gene or part gene form of a specific target gene normally found in that micro-organism, whereby upon infection of the microorganism by the vector and expression of the antisense gene or part gene form of that target gene in the host, translation of that gene is prevented or inhibited and the properties and/or characteristics of the micro-organism thereby modified.
  • a suitable vector type for use in accordance with this invention is a bacteriophage.
  • the natural action of bacteriophage is to infect the host cells with their own DNA to be replicated by the hosts biochemical machinery. Some bacteriophage act to lyse the host cell thereby destroying it. Others often lie in a lysogenic state in which they are normally of no significant detriment to the host.
  • the present invention provides for the genetic engineering of bacteriophage (phage) to introduce thereto an antisense gene or part gene form of a gene normally found in the micro-organism specifically targeted for modification.
  • phage bacteriophage
  • One of the advantages of using antisense forms of normally occurring genes or part genes is that the construct should not be considered a Genetically Modified Organism (GMO) according to the standard definition used in current regulations surrounding GMO's. This should enable relative freedom to use the invention in both natural and industrial environments.
  • GMO Genetically Modified Organism
  • a phage containing an antisense gene sequence could be achieved in any number of ways.
  • the method outlined below has been shown to be an effective strategy. It is important to note that the details given are specific for double strand DNA phage - alternative molecular methods would need to be employed if the nucleic acid of the phage was composed of either single strand DNA or RNA but the principle of the approach would still be the same.
  • DNA is prepared from the phage and size estimation made using restriction enzyme analysis. It is important to use phage with a large enough genome so that there is sufficient room to accommodate the additional inserted sequences within the size constraints of the packaging mechanism; generally phage can only accommodate -105% of their native genome size within the capsid.
  • the restriction analysis also allows construction of a physical map of the phage genome that will aid the identification of suitable sites for insertion of the antisense DNA in the phage genome following analysis of patterns of gene expression.
  • This DNA is used to construct a gene library of the complete phage genome which is used for subsequent sequence analysis which is required to screen the phage genome for potentially harmful sequences (such as phage- encoded toxin genes). It is desirable to ensure that such sequences are not present in any phage that will be released into the environment.
  • the phage DNA fragments are used as probes to identify regions containing lytic gene functions by Northern hybridisation analysis of infected cells. Since it is desired that the antisense gene is constitutively expressed in the lysogenic state, it is best to avoid such late gene regions that will be organised into transcriptional units which are only highly expressed during the lytic development of the phage.
  • the ideal choice of insertion site is outside the late gene region (this approach also avoids disruption of any late gene functions required for the lytic cycle which must be retained intact) and situated in a region of the genome which clearly contains no gene coding regions. Without a full genetic analysis it is always difficult to ensure that the regions chosen by sequence analysis alone will not have some cryptic function, disruption of which will result in a nonfunctional phage. By choosing two or three potential sites and using a selection method based on a functional phage phenotype it will be possible to circumvent such problems.
  • the antisense form of the gene and/or part gene is coupled to a strong promoter which is known to be constitutively expressed in the target organism.
  • a DNA construct containing the antisense gene fused to the promoter is introduced into a cloned fragment of the phage genome at the site identified, such that the antisense gene sequence is flanked by at least 0.5 Kbp on each side by the phage genome sequences surrounding the point of insertion.
  • This gene arrangement is constructed in an Ecoli shuttle vector capable of replicating in both Ecoli and the target organism and once constructed, the antisense gene/phage DNA gene arrangement is introduced into the target bacterium.
  • the host strain containing the plasmid is infected with the wild type phage.
  • the infection is carried out under conditions which favour the lytic cycle and phage particles recovered from lysed cultures by standard methodologies.
  • the expectation is that the double homologous recombination event will occur at a frequency of 1 in 10 (> , although frequencies greater than this can be expected if shuttle vectors are used which replicate by a rolling circle (single-stranded DNA) mode of replication.
  • the phage recovered from the lysate will be used to infect a lawn of host bacteria so that plaques can be screened by plaque lift and DNA hybridisation techniques to detect the presence of the antisense gene sequences (this may require multiple rounds of iteration due to the presence of homologous sense strand sequences within the plaques resulting from lysis of the host cell and release of chromosomal DNA sequences).
  • This process will require a primary screening event covering a library of a minimum of 10° plaques.
  • Use of a biological screening approach will allow selection against those constructs which have produced a defective recombinant phage which is unable to carry out the lytic cycle of infection.
  • Putative positive phage isolates are purified and further DNA analysis performed to confirm that the antisense gene sequences have been correctly integrated. These phage are then used to infect a lawn of bacteria to ensure that lysogens can still be generated (again the use of a biological assay quickly allows screening of the effect of the gene insertion on phage gene function without requiring extensive genome analysis).
  • Lysogens identified are screened for a functional phenotype - i.e. repression of the gene targeted for control by the antisense technology. This assay will determine the effectiveness of the introduced antisense form of the gene or part gene preventing or inhibiting translation of the target gene to ensure that the antisense gene present in the phage genome is biologically functional.
  • a recombinant phage will have been recovered which contains a copy of the antisense gene, and yet retains functional lytic and lysogenic gene control circuits.
  • the small size of antisense gene sequences that can be used means that the technique is unlikely to be limited by phage packaging constraints even when antisense forms of a plurality of different genes and/or gene parts are inserted.
  • the introduction of native host sequences into the phage genome is also likely to be regarded as homologous cloning and therefore not be subject to the constrains of current genetic containment regulations for the purposes of release into the environment, as mentioned above.
  • the cells of target micro-organisms can be infected with the bacteriophage resulting in either the cells being lysed or the bacteriophage becoming dormant (lysogenic state).
  • the phage DNA enters the cell and becomes part of the host genome. Therefore when the bacteriophage becomes dormant the antisense gene construct becomes part of the host genome. This gene construct will be constitutively expressed because it is coupled to a strong promoter.
  • the mRNA transcribed from the antisense gene construct will tend to bind to the target gene sense mRNA forming a duplex which blocks the processing and translation of the sense mRNA thereby preventing or severely inhibiting the formation of the protein usually formed from the target genes whereby properties of the micro-organism involving the protein that would normally have been formed is modified.
  • Different antisense bacteriophages can be engineered to be effective in a wide range of applications. For example, bacteria which have become multi- resistant to antibiotics (e.g.
  • tuberculosis and Staphylococcus aureus may be modified by infecting them with bacteriophage containing one or more antisense forms of genes or part genes specific to target genes in the bacteria and involved in the bacteria's resistance thereby blocking or inhibiting the production of the protein(s) which confer the antibiotic resistance. Therefore the antibiotics would become effective treatments for these resistant bacteria again.
  • the invention could be incorporated into wound dressings as a prophylactic against infection.
  • the invention could also be used to make safe sites contaminated with undesirable biological agents involving bacteria or possibly other microorganisms by targeting proteins essential to the survival of the micro-organism.
  • Water courses may be treated with antisense bacteriophage according to the invention to control the expression of virulence determinants of pathogenic microbes, for example vibrio cholerae. All manner of natural and industrial environments such as food, pharmaceutical, dairy, paint and oil industries may also benefit from the application of this invention.
  • the usefulness of the above methodologies may be lessened if for example the bacteria which are targeted produce mutant versions of the genes of interest the translation of which is not prevented or inhibited by the use of antisense forms of the normal gene.
  • This problem can be overcome by targeting the bacteria in its normal environment where it does not cause disease or exhibit its other undesirable characteristics and where the target gene is not actively or specifically expressed. Therefore, the presence of the phage delivering the antisense form is not detrimental to the bacteria in its normal environment.
  • An example of the use of methodology of the present invention to prevent or inhibit translation of virulence genes that are not expressed in the normal environment of bacteria would be a phage that inhibits expression of the verotoxin gene of Ecoli 0157.
  • This gene product appears to play no significant role in adult cattle (the principal natural host of the organism). It should therefore be possible to inhibit this gene using a lysogenic phage delivering an antisense form of the gene in cattle without seriously affecting the survival of the organism in its natural environment. Thus, the strong selective pressure in favour of phage resistant variants would be avoided.
  • the inhibition of the verotoxin gene should have significant beneficial effects on the severity of the resulting infection. Selection for resistant variants will occur at this stage, but assuming the infection is transient, unlikely to cycle back into the cattle, and because the variants have no advantage even if they did, they should not come to dominate in the environment.
  • a further example would be the introduction of lysogenic phage into a human commensal organism such as S. aureus or N.meningitidis.
  • the phage would carry an inhibitor of a gene product required for virulence when the organism is introduced into a potentially harmful site such as a wound or the bloodstream.
  • Cholera in water courses could also be treated in this manner because there appears to be no selective pressure towards virulence factors such as cholera toxin when the cholera is present in the water.
  • lysogeny occurs in a non-selective environment as above, the bacteria are not removed from the population since the genes for anti-sense gene control do not give the bacteria either a selective growth advantage or disadvantage in that environment.
  • the lysogenic bacteria will be modified by the anti-sense gene and yet maintained in the population. It is only when the organism enters its host animal that the effect of the anti-sense gene will be apparent, leading to an alteration of virulence and a reduced likelihood of disease.
  • the present invention also includes a further method of modifying the properties of target micro-organisms and a vector and construct for use in such a method.
  • the method comprises introducing to a micro-organism to be modified, a vector comprising an antisense gene or part gene form of a target gene normally found in that micro-organism, whereby upon infection of the micro-organism by the vector the RNA transcribed from the antisense form targets the RNA of the target gene for degradation thereby preventing protein formation from the target gene.
  • the properties of the micro-organism are thereby modified.
  • Ribonuclease P is a ubiquitous enzyme that is responsible for 5' end processing of tRNA and 4.5S RNA in bacteria.
  • the enzyme is comprised of a catalytic RNA subunit and a single scaffold protein.
  • RNAseP uses a combination of structural and sequence cues to recognise its target RNA. It has been shown conclusively that RNAseP can be redirected to cleave other RNA molecules by providing it with a short RNA that has 11 or more bases complementary to the target RNA, followed by CCA at its 3' end. These short RNAs have been dubbed External Guide Sequences (EGS).
  • EGS External Guide Sequences
  • EGS Hybridisation of an EGS to its target RNA produces a duplex molecule that mimics the substrate of RNAse P and results in the catalytic cleavage of the target molecule.
  • EGSs efficiently inhibit expression of plasmid-borne beta-lactamase and chloramphenicol acetyl transferase genes, with the consequent restoration of drug sensitivity.
  • Fusion of EGS to the catalytic Ml RNA subunit of RNAseP (Ml EGS) has been shown to significantly increase the efficiency of target mRNA cleavage.
  • M1EGS targeting the bacteriophage Lambda N gene significantly reduced the burst size of the phage. Expression of EGS therefore provides an effective means of selectively inhibiting the expression of specific bacterial mRNAs.
  • EGS can inhibit gene expression by up to 80%. This level can be improved by using multiple EGS to the same target RNA, and determining the optimal, most accessible sites for cleavage in any particular mRNA.
  • EGS External Guide Sequences
  • the effectiveness of the EGS is initially determined by measuring the expression of the target genes in vitro. How the observed level of inhibition translates into decreased virulence is determined using an in vivo model system.
  • RDEC-H19 rabbit infection model The in vivo model chosen is the RDEC-H19 rabbit infection model.
  • RDEC- 1 is a naturally occurring enteropathogenic E.coli (EPEC) that causes disease in rabbits. After oro-gastric challenge it colonises the caecum and proximal colon and causes a self-sustained enteric infection with diarrhoea in 6-10 day old rabbits. Histologically it demonstrates the same "intimate association" and "attachment and effacement” lesions that are typical of EPEC infection in humans.
  • RDEC-H19A is an isogenic variant of RDEC-1 that was infected with the ⁇ H19A Slt-I converting bacteriophage.
  • This organism expresses the human Slt-I gene at levels comparable to the human HI 9 strain from which the gene originated. It causes a more severe disease in rabbits that closely resembles the EHEC-induced colitis in humans. It is considered to be the best small animal model for EPEC/EHEC infections of humans. RDEC-1 does not infect rabbits asymptomatically, so this model is not suitable for studying the effects of EGS inhibition of virulence factors in asymptomatic hosts such as adult cattle.
  • E.coli The pathological effects of enteropathogenic E.coli are dependent upon the expression of several virulence factors, most of which are contained within the region of Ecoli known as the Locus of Enterocyte Effacement (LEE).
  • LEE Locus of Enterocyte Effacement
  • This example targets factors that are highly conserved between human EPEC/EHEC isolates and the RDEC-H19 strain that are used as the model system.
  • RDEC-1 "knock out" mutants are available for all of these genes. Comparison of the knockout mutants to the "EGS knockouts" allows us to gauge the success of the EGSs at suppressing virulence.
  • EGS target sites are identified for both the human (E.coli 0157)- and rabbit (RDEC-l)-specific genes. The chosen genes are: 1.
  • Verotoxin (VT or Shiga-like toxin SLT) is the product of the slt-lA and B genes carried on a lambdoid bacteriophage. They are part of a single operon and are translated from the same mRNA. The operon is induced in response to iron starvation.
  • Intimin (eaeA) is an outer membrane protein that is required for the attachment and effacement phenotype. This is an essential process required for enteropathogenicity. Intimin binds to ...
  • Tir protein espE
  • espE a bacterial gene product that is translocated into the cytoplasm of colonised epithelial cells.
  • EspB (formerly eaeB) is also translocated into the eukaryotic target cell, and is necessary for insertion of the Tir protein into the cell membrane.
  • the versatility of the EGS technology means that it can readily be applied to any other virulence factor genes that are identified.
  • Some degree of replication of the phage is desirable, as this would facilitate spread of the phage throughout the gut flora, and allow infection of pathogens that colonise the host some time after it was inoculated with the phage. Because the infected bacteria survive, they do not leave a vacant niche for the uninfected pathogens (resistant variants or organisms to which the phage could not gain access) to supersede them. The anticipated long term result is the decrease in the prevalence of the virulent organisms and the consequent decrease in the probability of transmission of virulent organisms to humans.
  • the bacteriophage must be lysogenic, capable of infecting E.coli and be suitable for manipulation via recombinant DNA techniques. Lambda bacteriophage is used in this example because this is the best characterised and simplest to manipulate of all of the suitable phage.
  • EGS External Guide Sequences
  • an inducible system that only activates expression of the EGS at an appropriate time, for example when Verotoxin gene expression is activated. This could be achieved by using an inducible system based on the sit promoter under the control of the Fur repressor. Similarly, using an inducible promoter it should be possible to switch expression of the virulence gene off at will. However for the purposes of this example, a constitutive promoter is used.
  • EGS expression of multiple EGS is achieved either by using multiple promoters, or preferably by expressing the EGS as tandem copies separated by short hammerhead ribozyme sequences that will result in autocatalytic processing of the RNA into the individual EGS.
  • the promoter/EGS/terminator cassette(s) are assembled using PCR based strategies in such a way that they will be bounded by unique BamHI and Hindlll sites. This facilitates their subsequent cloning into the Lambda vector described below.
  • the multiple EGS expression plasmids are electroporated into E.coli. Expression, and where applicable, processing of the EGS is confirmed by Northern blot and ribonuclease protection assay. The plasmids are then tested to determine their effect on expression of the virulence genes themselves in terms of steady state mRNA level using ribonuclease protection assays; the level of protein expression using immunoprecipitation and Western Blot assays; and activity using biological assays of gene function.
  • EGS EGS on virulence in vivo
  • Groups of 3-5 rabbits are infected with 10 ⁇ ' cfu of RDEC-H19 carrying the EGS expression plasmids for eaeA, espB, espE and slt-I.
  • Controls include RDEC-H19 carrying a plasmid that expresses an irrelevant set of EGS and RDEC-1.
  • Drinking water containing an appropriate antibiotic is used to minimise plasmid loss.
  • Stools are examined daily and noted for the presence and severity of diarrhoea.
  • RDEC- H19 is Nal r
  • the proportion of bacteria that have lost the EGS plasmid is determined using plates containing Nal alone. 7 days post infection the rabbits will be killed and the jejunum, ileum, caecum, proximal and distal colons examined histologically for lesions using established criteria for enteroadherance, mucosal changes, heterophilic infiltration, oedema, and vascular changes. 11-1 content of the mucosal tissue is assayed as a quantitative measure for mucosal inflammation.
  • the target that proves to be most sensitive to EGS inhibition and beneficial in terms of reduced virulence is carried through to the second phase, the construction of bacteriophage that deliver the EGS expression cassette and disseminate it throughout a population or organisms.
  • Lambda DNA is purified from phage particles and digested with restriction enzymes and subcloned such that the resultant plasmid will have unique restriction sites that will permit the insertion of the EGS expression cassette described above.
  • the subcloned engineered sequence can then be reinstated into its original position in the phage genome. These replace approximately 800 base-pairs corresponding to the C-terminal regions of the non-essential genes of the Lambda DNA. Transcription from the EGS cassette is in the same direction as for Lambda pL early genes to avoid potential antisense inhibition early gene expression resulting from readthrough of the EGS terminator.
  • the ligation is directly electroporated into Ecoli and the resultant turbid plaques screened by nucleic acid hybridisation and confirmed by restriction analysis.
  • the sequence of the recombinant phage is confirmed by sequencing using dye-labelled primers either side of the BamHI and Hindlll sites.
  • the phage is plaque cloned a second time, and checked by hybridisation to ensure that they do not carry any inappropriate sequences.
  • mutants Because of the large number of defined mutants that are available for Lambda, it is possible to fine-tune the behaviour of the phage. For example, mutants could be generated that have enhanced tendencies to become lysogenic, or possess different immunity regions to broaden or limit their potential host range.
  • phage vector to infect, lysogenise and reactivate from RDEC-1 is then confirmed in in vitro culture.
  • the effectiveness of virulence gene inhibition in lysogenised bacteria is determined using in vitro assays as described for the plasmid borne EGSs above. After a significant reduction in virulence gene expression has been demonstrated, a series of rabbit infection experiments are performed to ensure the effectiveness of the phage vector.
  • a vector comprising an antisense form of a target gene normally found in a micro-organism to target the RNA translated from the target gene for degradation via the action of Ribonuclease P. Therefore translation of the target gene is prevented and thereby the undesirable properties or characteristics associated with the protein(s) expressed from the gene are attenuated.
  • the methodologies and vectors of the present invention may also be used to target specific genes for sequence specific cleavage of mRNA's by ribozymes thereby inhibiting gene expression as required.
  • a plurality of antisense gene or part gene forms specific to target genes in one or more micro-organisms may be coupled to a common promoter and inserted into a single bacteriophage thereby enabling more than one gene in a given or a plurality of target micro-organisms to be targeted.

Abstract

L'invention concerne des procédés de modification de micro-organismes, permettant particulièrement de contrôler les micro-organismes. Ces procédés consistent à introduire dans un micro-organisme cible un vecteur comprenant une forme antisens de la partie entière d'un ou plusieurs gènes cible présents normalement dans ce micro-organisme. Après infection du micro-organisme par le vecteur et l'expression de la forme antisens de ce gène ou d'une partie de ce gène dans l'hôte, on évite ou inhibe la traduction du gène cible et les propriétés des micro-organismes modifiés ce qui garantit le contrôle des micro-organismes.
PCT/GB2000/004795 1999-12-17 2000-12-15 Modification de micro-organismes WO2001044456A2 (fr)

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DE102007003148A1 (de) * 2007-01-15 2008-07-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Gentechnisch modifizierte Bakteriophagen, insbesondere zur Bekämpfung von pathogenen Prokaryonten bzw. ihrer pathologischen Wirkung, sowie deren Verwendung und Herstellung

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US5552144A (en) * 1992-01-22 1996-09-03 Microcarb, Inc. Immunogenic shiga-like toxin II variant mutants
US5821052A (en) * 1992-04-16 1998-10-13 University Of Medicine And Dentistry Of New Jersey Control of the synthesis of proteins by anitisense RNA-tRNA complex
WO1999027135A2 (fr) * 1997-11-21 1999-06-03 Yale University Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules

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US5552144A (en) * 1992-01-22 1996-09-03 Microcarb, Inc. Immunogenic shiga-like toxin II variant mutants
US5821052A (en) * 1992-04-16 1998-10-13 University Of Medicine And Dentistry Of New Jersey Control of the synthesis of proteins by anitisense RNA-tRNA complex
CA2078716A1 (fr) * 1992-09-21 1994-03-22 Joyce De Azavedo Proteine de e. coli enterohemorragique de fixation et de destruction
WO1999027135A2 (fr) * 1997-11-21 1999-06-03 Yale University Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules

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HARTH G ET AL: "Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L- glutamate/glutamine cell wall structure, and bacterial replication." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (2000 JAN 4) 97 (1) 418-23. , XP002167663 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007003148A1 (de) * 2007-01-15 2008-07-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Gentechnisch modifizierte Bakteriophagen, insbesondere zur Bekämpfung von pathogenen Prokaryonten bzw. ihrer pathologischen Wirkung, sowie deren Verwendung und Herstellung

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WO2001044456A3 (fr) 2001-12-27
AU2196601A (en) 2001-06-25

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