WO2002079467A2 - Selection de souche bacterienne sans antibiotique a l'aide de molecules antisens - Google Patents

Selection de souche bacterienne sans antibiotique a l'aide de molecules antisens Download PDF

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WO2002079467A2
WO2002079467A2 PCT/DK2002/000208 DK0200208W WO02079467A2 WO 2002079467 A2 WO2002079467 A2 WO 2002079467A2 DK 0200208 W DK0200208 W DK 0200208W WO 02079467 A2 WO02079467 A2 WO 02079467A2
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pna
gene
antisense
seq
cells
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WO2002079467A3 (fr
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Peter E. Nielsen
Liam Good
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Københavns Universitet
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    • 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
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3183—Diol linkers, e.g. glycols or propanediols
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • the present invention relates to a new method for an antibiotic-free selection of genetically modified cells.
  • the method is based on the combination of antisense molecules, e.g. peptide nucleic acid (PNA) reagents, that are targeted against a growth essential cellular gene and the transformation of the cells with a recombinant DNA- cloning vector coding for a functional, but mutated version of said growth essential gene that is refractive to the antisense molecule.
  • PNA peptide nucleic acid
  • a basic and essential requirement for effective genetic engineering of bacteria and other cells propagated in cell cultures is the capacity to select for a cell with a specific genotypic alteration.
  • the most common selection strategy in recombinant DNA technology is to include a cloned gene or DNA sequence in a genetic element (plasmid, virus, transposon etc.) that has a phenotypical property which allows for the separation of host cells containing the element (transformed cells) from cells that do not.
  • a selection of cells containing the wanted new genetic element can conveniently be achieved by growing cells on a medium containing a toxic substance and on which only the transformants expressing the "resistance gene" are able to survive.
  • genes are available that provide for virus resistance, heavy metal resistance or polypeptide resistance, most current protocols for the selection of transformed cells are based on genetic elements (e.g. cloning vectors) that express genes coding for antibiotic resistance in the transformed host cell (see e.g. US 4,237,224; Ausubel, 2000).
  • Illustrative antibiotics include penicillin tetracycline, streptomycin and sulfa drugs.
  • Antisense RNA regulation is a natural mechanism for gene regulation, occurring in a wide range of organisms wherein an antisense RNA binds and inactivates a target mRNA molecule (Wagner & Simons, 1994).
  • PNA Peptide nucleic acid
  • PNA compounds are an antisense compound that has shown good results for specific gene targeting in a number of different organisms (Good & Nielsen, 1997; Larsen et al., 1999; Nielsen et al., 1991, WO 93/12129, US 5,773,571).
  • the sugar backbone of an oligonucleotide is replaced with an amide containing backbone, in particular with an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that teach the preparation of PNA compounds include U.S. Pat. Nos.
  • PNA has successfully been targeted against both reporter genes and an essential gene (Good & Nielsen, 1998a; Good & Nielsen, 1998b). Furthermore, PNA molecules have shown good stability and high binding affinity, probably due to their uncharged pseudo- peptide backbone ( Figure 1). As no general toxicity has been detected so far, PNA constitutes a good candidate for use as an antimicrobial drug, as taught by WO 99/13893. A major difficulty, though, has been the inadequate rate of uptake of PNA into bacterial cells. However, the uncharged and hydrophilic properties of PNA, as well as the peptide backbone have been helpful in improving cellular uptake. For example, it has been possible to link specific carrier peptides to the PNA backbone so that membrane passage in eucaryotic cells was facilitated (Pooga et al. (1998) Nat Biotechnol 16, 857-61). Summary of the invention
  • the present invention addresses the above stated complications by providing a substantially new and inventive method for selecting for a transformed cell without using antibiotics.
  • the invention is based on the observation that it is possible to design antisense molecules that specifically inhibit the expression of an essential cellular gene, but which do not inhibit the expression of a mutated, but functional homologous version of the same gene.
  • the mutated, but functional version of the essential gene is incorporated into a cloning vector and the vector is introduced into cells, cells containing the vector will survive treatment with the antisense molecule, because the mutated, but functional homologous version of the essential gene will provide the essential function to the cells, whereas cells that do not contain the vector will die.
  • antisense molecules to select for transformed or transfected cells without the common use of antibiotics.
  • the antisense molecules are short antisense molecules that are able to down modulate bacterial gene expression both in vitro and/or In vivo.
  • the short antisense molecules are additionally complexed with cell wall or cell membrane transport peptides, thus improving the uptake of the antisense molecule into the cell.
  • antisense peptide nucleic acids are covalently conjugated with cell wall or cell membrane transport peptides.
  • PNA antisense peptide nucleic acids
  • Such peptide- PNA conjugates directed against various essential genes of Escherichia coll (E. coli) are herein shown to be able to inhibit bacterial growth both of permeable E. coli strains, such as the AS19 strain, and of E. coli K-12 strains that are wildtype with respect to permeability, as well as of Bacillus subtillis.
  • an antisense molecule directed against the essential acpP gene encoding the acetyl carrier protein Acp, which is central for fatty acid biosynthesis in E. coli (Cronan, 1996) can be used as a reagent with good antibacterial effects.
  • antisense PNA targeted against the essential gene acpP is used to select transformed E. coli cells which contain a plasmid carrying an altered acpP gene, in a mixed population consisting of both transformed and non- transformed cells.
  • acpP mRNA-targeted PNA as a selective agent for an E. coli cell that is transformed with a genetic element carrying a mutated, but functionally homologous version of said gene, was furthermore demonstrated by selection of plasmid carrying cells after electrotransformation.
  • the results show that antisense PNA can substitute the use of antibiotics and other environmentally problematic substances as selective agents in gene cloning procedures.
  • the invention provides several properties to microbial selection systems, as well as to other non-microbial selection systems, as described in the prior art.
  • the possibility to select and maintain a plasmid containing bacterial culture without the use of antibiotics can be used for a wide variety of applications within the fields of research, development and industrial production, involving genetically modified micro-organisms.
  • Another important aspect of the present invention is the use of embodiments of the present invention for inhibiting bacterial infections in eukaryotic cell cultures.
  • Yet another important aspect of the present invention is to use antisense constructs to "purify" or "decontaminate" microbial cultures used in industrial applications. In the diary and brewing industry it is of paramount importance to initiate and retain fermentation free of contaminating microorganisms.
  • the present intention relates to a novel method for antibiotic-free selection of transformed cells that is based on antisense molecules targeted against essential cellular genes.
  • Antibiotic-free selection in the present application refers to a situation wherein an efficient selection is obtained without use of antibiotics, such as tetracycline or ampicillin.
  • the present invention thus provides a method for selecting "genetically modified cells", wherein the selection is performed by modifying cells containing a growth essential gene X, inserting in such cells a "construct" containing a gene Y that is a mutated, but functionally homologous version of gene X (X 1 ), and treating said modified cells with an antisense or antigene construct directed against said essential gene X of the cells, thereby obtaining a preferential growth of the modified cells over other, non-modified cells.
  • genetic element generally means a polynucleotide comprising a region that encodes a polypeptide, part of a polypeptide, or a region that regulates transcription or translation or other processes important to the expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
  • Genetic elements comprise a polynucleotide molecule that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be described as plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), or a nucleic acid sequence that behaves as an autonomous unit of polynucleotide replication within a cell.
  • cloning vectors are the so-called "cloning vectors”.
  • a "cloning vector” or “vector” is herein defined as a plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), or nucleic acid sequence which is able to replicate in a host cell, characterized by one or a small number of restriction endonuclease recognition sites at which the sequence may be cut in a predetermined fashion, and which may contain an optional marker suitable for use in the identification of the cells that have taken up the vector, i.e. the "transformed” cells, e.g., tetracycline resistance or ampicillin resistance.
  • cloning vector or “vector” is the specific type of nucleic acid sequence that is not able to replicate in the target or final host cells but which - under selective pressure - can be inserted into chromosomes and are thereafter maintained without selective pressure. Often such non- autonomously replicating or nucleic acid sequences contain or are part of vectors that contain sequences allowing them to replicate in a suitable secondary host cell.
  • a cloning vector may or may not possess the features necessary for it to operate as an "expression vector” i.e. a vector that facilitates the transcription and/or translation of an appropriately inserted gene.
  • construct refers to a specific cloning vector with one or more specific polynucleotide fragments inserted in an ordered fashion.
  • a “growth essential gene” is herein defined as a gene whose inactivation is lethal to the organism, or alternatively, whose inactivation will inhibit or disturb the natural proliferation rate of an organism.
  • Antisense molecules or “antisense reagents” can, in the present context, be any molecule that hybridizes by a sequence specific base pairing to a complementary DNA and/or RNA sequence.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays, and in the case of in vitro assays, under conditions in which the assays are performed.
  • Typical antisense molecules or “antisense reagents” are any oligonucleotide, such as DNA, RNA, any peptide nucleic acid, any other nucleic acid derivative, or mimic and/or derivative thereof.
  • the target sequence is not restricted to the “sense” or “coding” strand of mRNA, although this is often the target.
  • the present invention employs "antisense compounds", or “antisense constructs” which are used interchangeably in the present text.
  • PNA and oligonucleotides for use in modulating the function of nucleic acid molecules encoding essential genes is addressed. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding said essential gene.
  • target nucleic acid and “nucleic acid encoding said essential gene” encompass a DNA encoding said gene, and/or an RNA (including pre-mRNA and mRNA) transcribed from such DNA.
  • RNA including pre-mRNA and mRNA
  • the specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
  • This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense” (when the target is RNA) or “antigene” (when the target is DNA).
  • antisense when the target is RNA
  • antigene when the target is DNA
  • the functions of DNA to be interfered with include replication and transcription. This effect is referred to as "antigene”.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA and is referred to as "antisense".
  • antisense and “antigene” is not absolute.
  • the overall effect of such interferences with target nucleic acid function is a specific modulation of the expression of said essential gene.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • inhibition is the preferred form of modulation of gene expression.
  • Gene Y on the vector is ideally able to complement the essential cellular gene X.
  • a "complementing" gene is a gene that by its presence is able to restore the phenotype of a mutated cell. To restore the phenotype of a mutated cell, the complementing gene does not need to be identical to an unmutated version of said gene.
  • One particular example is the so-called alpha- complementation of the E. coli lacZ gene.
  • the mutated gene and the mutated complementing gene are mutated in different positions, the result being a restoration of the function of the non-mutated gene through complementation.
  • the essential cellular wild-type gene is not mutated but inactivated through the specific interaction of the antisense construct.
  • the viable phenotype is restored by the presense of an altered but yet functionally equivalent version of said essential gene.
  • the altered version of the essential gene is sufficiently different in nucleotide sequence to be refractive to the inhibitory effect of the antisense construct.
  • gene Y on the vector is a mutated but functionally equivalent of the essential cellular gene X, it is referred to as X'.
  • the mutated gene X' on the vector can e.g. be a "silent mutation" of gene X.
  • a "silent mutation” is a mutation which does not lead to a change in the product encoded by the gene.
  • antisense molecules can be selected from the group consisting of oligonucleotides, oligonucleotide analogues, oligonucleotide mimics, such as for example PNA, locked nucleic acids (LNA), phosphorothioate, 2'-methoxy-, 2'-methoxyethoxy-, morpholino, phosphoramidate oligonucleotides or the like.
  • antigene molecules can furthermore be selected from the group consisting of triplex forming or strand invading oligonucleotides, oligonucleotide analogues, oligonucleotide mimics, such as for example PNA, locked nucleic acids (LNA), phosphorothioate, 2'- methoxy-, 2'-methoxyetyhoxy-, morpholino, phosphoramidate oligonucleotides or DNA minor groove binding polyamides (oligo pyrroles/imidazoles etc.) as described (Gottesfeld JM et al. 2000; Dervan PB et al. 1999) or the like.
  • PNA locked nucleic acids
  • LNA locked nucleic acids
  • phosphorothioate 2'- methoxy-, 2'-methoxyetyhoxy-
  • morpholino phosphoramidate oligonucleotides or DNA minor groove binding polyamides (oligo
  • oligonucleotide(s) refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages which function similarly or combinations thereof.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases and other enzymes, and are in the present context described by the terms "oligonucleotide analogues" or "oligonucleotide mimics”.
  • the present invention also comprises other oligomeric antisense compounds, including, but not limited to, oligonucleotide mimetics such as described below.
  • the antisense compounds in accordance with this invention preferably comprise from 4 to 30 nucleobase units.
  • nucleobase units is used in the present text to describe both the number of nucleotides in a oligonucleotide and the number of nucleobase-carrying monomers of a oligonucleotide mimetic.
  • Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from 7 to 15 nucleobases.
  • a nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5 1 hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure; however, open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorous atom in the backbone and those that do not have a phosphorous atom in the backbone.
  • modified oligonucleotides that do not have a phosphorous atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphospho- triesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogues of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified oligonucleotide backbones that do not include a phosphorous atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones consist of oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular ⁇ CH 2 --NH-- O— CH 2 --, ⁇ CH 2 --N(CH 3 )--0--CH 2 ⁇ [known as a methylene (methylimino) or MMI backbone], ⁇ CH 2 ⁇ 0 ⁇ N(CH 3 ) ⁇ CH 2 --, -CH 2 --N(CH 3 )--N(CH 3 )--CH 2 - and -0 ⁇ N(CH 3 )--- CH 2 — CH 2 — [wherein the native phosphodiester backbone is represented as ⁇ 0--P--0-- CH 2 — ] of the above referenced U.S.
  • modifications include 2'-methoxy (2'-0-CH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotides. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substi
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. (Sanghvi, Y. S., Crooke, S. T. and 10 Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
  • modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777- 3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &
  • oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular ⁇ CH 2 --NH-- 0 ⁇ CH 2 --, — CH 2 ⁇ N(CH 3 ) ⁇ O— CH 2 ⁇ [known as a methylene (methylimino) or MMI backbone], ⁇ CH 2 --0 ⁇ N(CH 3 )-CH 2 ⁇ , ⁇ CH 2 ⁇ N(CH 3 )--N(CH 3 )-CH 2 ⁇ and ⁇ 0--N(CH 3 ) ⁇ CH 2 ⁇ CH 2 ⁇ [wherein the native phosphodiester backbone is represented as --0--P-- 0 ⁇ CH 2 --] of the above referenced U.S.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O—, S— , or N-alkyl; 0 ⁇ , S— , or N-alkenyl; 0 ⁇ , S ⁇ or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 3 to C 3 0 alkyl or C 2 to C 3 0 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2' position: C 3 to C 3 0 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl group.
  • a preferred modification includes 2'-methoxyethoxy (2' ⁇ 0 ⁇ CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Heiv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • Other preferred modifications include 2'-methoxy (2'-0-CH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F).
  • oligonucleotide Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • Preferred embodiments of the invention are oligonucleotide mimetics, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an
  • PNA peptide nucleic acid
  • linker in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in
  • PNA protein nucleic acid
  • PNA any peptide or peptide conjugated molecule comprising one or more PNA monomers. Also molecules containing fully or partially modified PNA are considered PNA.
  • the oligonucleotide mimetics are a bi-cyclic DNA analogue called Locked Nucleic Acid (LNA) (see International Patent Application WO 99/14226; P. Nielsen et al, J. Chem. Soc, Perkin Trans. 1, 1997, 3423; P. Nielsen et al., Chem. Commun., 1997, 9, 825; N. K. Christensen et al., J. Am. Chem. Soc, 1998, 120,
  • LNA Locked Nucleic Acid
  • LNA in the present context means any oligonucleotide either fully or partially modified with LNA monomers.
  • LNA monomers any monomeric unit is meant that comprises the LNA bi-cyclic structure.
  • a preferred embodiment of the present invention thus relates to a method for selecting genetically modified cells, wherein the selection is performed by modifying cells containing a growth essential gene X with a construct containing a gene Y, and treating said modified cells with an antisense or antigene construct directed against said essential gene X of the cells, thereby obtaining a preferential growth of the modified cells over other, non-modified cells,
  • a particularly preferred embodiment of the present invention thus relates to a method for selecting genetically modified cells, wherein the selection is performed by modifying cells containing a growth essential gene X with a construct containing a gene Y, and treating said modified ceils with an antisense or antigene construct directed against said essential gene X of the cells, thereby obtaining a preferential growth of the modified cells over other, non-modified cells, wherein said antisense construct is a PNA.
  • Said antisense construct can additionally be a modified PNA, wherein the modification is e.g. obtained by linking the PNA to a peptide, a carbohydrate or a lipid or wherein the modification is obtained by internal modification, i.e. backbone modification, in which the glycine of the PNA backbone is exchanged for other ⁇ -amino acids.
  • the PNA is covalently linked to the KFFKFFKFFK peptide.
  • the presence of the KFFKFFKFFK (SEQ ID NO: 1) peptide substantially increases the efficacy of the PNA-antisense construct.
  • Antisense PNAs are larger than most drugs, e.g. antiobiotics. Thus PNA size or length is likely to be an important parameter for the permeabilizing and thus the efficacy.
  • PNAs targeted to the start codon region of the chromosomal ⁇ -galactosidase gene (lacZ) (see Figure 9 and Table 5) were synthesized over the 7 to 15 mer size range. Inhibition of lacZ expression was determined using an E. coli liquid culture assay (for details see Good and Nielsen 1998a and 1998b, Good et al. 2001). The results are illustrated for a wild type E.
  • an especially preferred embodiment of the present invention method relates to a method for selecting genetically modified cells, wherein the selection is performed by modifying cells containing a growth essential gene X with a construct containing a gene Y, and treating said modified cells with an antisense or antigene construct directed against said essential gene X of the cells, thereby obtaining a preferential growth of the modified cells over other, non-modified cells, and wherein said antisense construct contains a PNA consisting of 4-30 nucleobase units, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleobase units.
  • antisense construct that contains a PNA consisting of 10-12 nucleobases
  • said antisense construct contains a PNA consisting of 7-15 nucleobase units.
  • PNA protein adenosine nucleic acid
  • eucaryotic HeLa cell cultures were infected with 10 4 colony forming units (cfu) of wild type E. coli K 12 and antisense peptide-PNA conjugate directed against the acpP of E coli was added.
  • cfu colony forming units
  • PNA at a concentration of 2 ⁇ M or higher fully cured the HeLa culture of E coli infection, no visible effect was seen on the HeLa cells even at the highest concentration tested (20 ⁇ M).
  • E. coli outer cell wall is a major barrier to PNA (Nikaido, 1994).
  • bacteria are permeabilized by cationic antimicrobial peptides and such compounds can act synergistically with antimicrobials that enter cells poorly (Hancock, 1997).
  • E. coli cells permeabilized by the polymyxin nonapeptide are more susceptible to PNAs. Covalent attachment of such a permeabilizing agent can further improve cell entry, as has been demonstrated in related applications with eukaryotic cells (Schwarze, 2000; Aldrian-Herrada, 1998; Pooga, 1998; Cutrona, 2000).
  • KFFKFFKFFK a quite simple synthetic peptide (SEQ ID NO: 1)) was recently shown by Vaara (1996) to facilitate uptake of antibiotics into bacteria without any inherent antimicrobial activity.
  • Vaara a cell wall permeating peptide
  • 12 and 15 mer anti-/acZ PNAs were synthesized with a KFFKFFKFFK (SEQ ID NO: 1) peptide attached via a flexible ethylene glycol linker (Table 5).
  • lacZ antisense constructs can be an antisense molecule, such as a PNA, that is covalently linked to a cell wall or cell membrane transport peptide to improve its uptake into a cell.
  • cell wall or cell membrane transport peptide are peptides comprising the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 1) or KFFKFFKFFKC (SEQ ID NO: 2) or IKFLKFLKFLC (SEQ ID NO: 3) or VDKGSYLPRPTPPRPIYNC (SEQ ID NO: 4) or ILPWKWPWWPWRRGC (SEQ ID NO: 5) or KLAKALKKLLC (SEQ ID NO: 6).
  • the antisense construct is a PNA covalently linked to a cell wall or cell membrane transport peptide comprising the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 1).
  • the present invention also includes antisense compounds which are chimeric compounds.
  • "Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids.
  • the antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • the present invention also provides a method for selecting for transformed cells that employs antisense molecules directed to the site of the start codon of an essential prokaryotic or eukaryotic gene X, and wherein the result of the interaction of said antisense with the mRNA of said essential gene is an inhibition of the translation of said essential gene X.
  • start codon or "initiation codon” are herein used interchangeably.
  • the start codon is the first mRNA triplet to be translated during protein or peptide synthesis and immediately precedes the structural gene being translated.
  • the start codon is usually AUG, but may sometimes also be GUG or UUG.
  • An essential gene of the present invention is typically selected from, but not limited to, the group of genes consisting of essential genes of Esche chia species and subspecies including E. coli and E. coll K12, Bacillus species and subspecies including ⁇ . subtilis, Saccharomyces species including S. cerevisiae and S. carlsbergensis,
  • lactis Pichia species including Pichia pasto s, Hansenula species including Hansenula polymorpha, Yarowia, Schwaniomyces and Zygosaccharomyces species.
  • Most of these organisms are of major industrial value as persons skilled in the art will know. Many of the organisms find important use in the industrial production or processing of food. Within this field of industrial activity, it is of paramount importance that the level of antibiotics used during the activity is extremely low or nil.
  • the present invention offers an efficient solution to the problem of producing food using genetically modified organisms, while leaving very little or no trace of antibiotics in the product.
  • An essential gene X is therefore preferably selected from, but not limited to, the group of genes consisting of E. coli genes: acpP, accD, acpS, frr, infA, Int, murA, murl, parC, proS, rpoE and rpsB, and/or Bacillus subtilis genes acpA, sigH, nadE, sbp, ftsZ, sipS, sigA, tagH and tagG and/or Saccharomyces cerevisiae genes ACC1,T0P2, ALA1, CDC4, CDC28, RPC25, PMI40, PAP1, TFC8, YKT6, TAF145 and TIF11.
  • a method for selecting genetically modified E. coli cells wherein said modified cells are treated with an antisense or antigene construct directed against the essential acpP gene of E.coli, and wherein said antisense or antigene construct reduces the level of expression of the growth essential acpP gene, thereby excercising a bacterioicidal and/or bacteriostatic effect on the untransformed cells in the mixed culture.
  • the antisense construct can for instance comprise the nucleobase sequence: (5'-/N-)
  • CTCATACTCT(3'-/C-) as shown in SEQ ID NO: 7 or (5'-/N-)CATACTCTTAAA(3'-/C-) shown in SEQ ID NO: 8, or (5'-/N-)CCTATCAAAACT(3'-/C-) as seen in SEQ ID NO: 9.
  • Gene Y on the vector is ideally able to complement the cellular gene X because gene Y is a mutated, but functionally equivalent version of gene X, herein defined as X'.
  • gene X 1 typically stands for a gene coding for the wild-type product of gene X, such as for the acpP gene, which is mutated at one or more positions between the Shine- Delgarno sequence (ribosome binding site) and the start codon of the gene.
  • a gene X' comprises the sequence S'-AACAGAATTCATGAGCACTA-S' (SEQ ID NO: 19).
  • the present inventors have previously shown that a triplex-forming PNA oligomer (PNA 1143) targeted to the ⁇ -sarcin loop of the 23S ribosomal RNA inhibits the growth of E. coli. (Good, 1998).
  • the attached peptide was shown also to improve the potency of such antimicrobial PNAs, when a KFFKFFKFFK (SEQ ID NO: 1) peptide was conjugated to this PNA.
  • the "naked" version of this anti-ribosomal PNA inhibited translation but only affected cell growth of a permeable E coli strain (AS19) in diluted growth media.
  • the new anti-ribosomal peptide-PNA conjugate (PNA1934) exhibited an MIC of 3 ⁇ M against E coli K12 grown in Mueller Hinton broth and an MIC of 700 nM when grown in dilute broth (Table 5). Again, free peptide and PNA 1143 were not inhibitory at these concentrations, showing that conjugation of the peptide with PNA is needed for potent growth inhibitory effects.
  • the potential for PNAs targeted against mRNA was shown as exemplified by a PNA complementary to the start codon region of the acpP gene.
  • This anti- acpP peptide-PNA conjugate (SP4) showed an MIC against E coli K12 of 1 ⁇ M in rich broth and an even lower MIC in dilute broth ( Figure 12A, Table 5).
  • Antisense specificity of the anti-acpP peptide-PNA was demonstrated in two ways. First, a control peptide-PNA with a scrambled PNA sequence (SP181, SEQ ID NO: 51) showed 15 fold higher MIC than the fully matched version SP4 (Table 5). This was consistent with an acpP specific inhibitory effect for SP4.
  • an anti-acpP PNA construct was tested against E. coli grown in cell culture medium.
  • HeLa cell cultures were infected with 10 4 cfu of non-invasive E. coli K12 and PNA was added immediately post infection. This system can be viewed as a model for the growth of an extracellular pathogen in a host.
  • the PNA did not visibly affect HeLa cell growth at the highest concentration tested (20 ⁇ M) ( Figure 14), and PNA at 2 ⁇ M or higher appeared to fully cure the HeLa culture of the infection. No live bacteria were detected from these samples in an agar colony forming unit test, confirming that the anti-acpP PNA is bactericidal under these conditions. Again, the sequence specificity of the effect was evaluated in cultures infected with E.coli carrying the binding-site-altered acpP allele (K12-acpP-I) and this infection was resistant to PNA up to at least 20 ⁇ M ( Figure 14).
  • Antisense PNAs are larger than most antimicrobials and the conjugation of a peptide carrier further increases the molecular weight to approximately 4000. Therefore, a 2 ⁇ M treatment corresponds approximately to 8 ⁇ g/ml, which is somewhat high compared to conventional antibiotics. Nevertheless, the observed antimicrobial effects within a cell culture system broaden the potential scope for the approach and raise the prospects for PNA-based antibacterial drugs.
  • nanomolar concentrations of PNA directed towards the essential E coli acpP gene not only inhibited bacterial growth, but also had a strong bactericidal effect. This shows that it is possible to replace antibiotic use with PNA. Similarly, it is demonstrated that PNA directed towards the Bacillus subtilis acpP homologue infer complete growth arrest and strong bactericidal effects (example 10). This results open possibilities to use antisense PNAs for strain selection and construction in research and for industrial applications in a wide range of bacteria.
  • FIG. 3 is a graphical representation of this experiment. Selection was carried out using a 1 ⁇ M concentration of anti-acpP PNA (PNA SP4). The results shown in Table 2 indicate a very good selective effect upon cells in a mixed culture. A strong selection effect is seen after just one hour (83.3% Amp r clones as compared to 50% in the start culture) and 100% selection efficiency was obtained within only two hours. As shown before, a strong effect of PNA upon wild type K12 cells is seen when grown as a monoculture. The rescue effect previously reported is also seen for the K12/acpP-l strain grown with 1 ⁇ M PNA.
  • Electroporation was chosen as the transformation method, and electrocompetent E. coli DH5 ⁇ were used. About 10 8 bacteria were transformed with 30 ng pKK223-3/ acpP-1 DNA. After recovery in SOC media for 40 minutes and dilution in 10% LB, low salt selection was carried out with a 1 ⁇ M concentration of wild type acpP-targeted PNA. Within 6 hours (see Table 3) the selection efficiency was almost complete (96.6% Amp r ), and after 18 hours the culture contained only ampicillin resistant bacteria.
  • FIG. 1 Chemical structure of DNA and PNA.
  • PNA the phosphodiester backbone is replaced with a pseudo-peptide backbone, which provides similar spacing between the bases (marked as b) as in DNA.
  • Hybridisation of antisense PNA oligonucleotides results in a stable helical complex and follow Watson-Crick pairing rules.
  • Anti-acpP PNA complementary to wild type and mutant acpP mRNA The structure of the wild type chromosomal and mutant plasmid-borne copies of the acpP genes are shown, along with the antisense PNA, which is complementary only to the native acpP mRNA start codon. Cells harbouring the mutant acpP allele are not affected by the presence of PNA whereas growth is arrested for wild type E. coli (as shown in Good et al. in press).
  • the harvested cells were diluted in water to different concentrations and 50 ⁇ l of each dilution was plated out on LB-plates.
  • the LB-plates were incubated at 37°C for 48 hours and an estimation of the total number of cfu was performed by counting the colonies formed. Colonies derived from each harvest time were transferred one by one to LBA-plates that were then incubated for 24 hours at 37°C. Only cells containing pKK223- 3/acpP-l would be able to grow since the plasmid contains the ampicillin resistance gene. The number of growing clones were counted and related to the total number of transferred clones (Table 2). After electrotransformation of E.
  • Plasmids were purified from colonies that grew on LBA-plates. The samples were loaded on a 1% agarose gel. Lanes 1 and 14 contain the marker ⁇ Hind III, lanes 2-7 and 9-13 contain samples derived from LBA- growing colonies, and lane 8 is a control with plasmid pKK223-3 acpP-1. Figure 5.
  • Map of plasmid pKK223-acpP-l Map of plasmid pKK223-acpP-l.
  • the insert shown at the top indicates the cloning strategy, construction and the sequence that replaces the native acpP start codon region (see also Figure 2).
  • the arrows indicate antibiotic resistance markers, the Ptac promoter and the origin or replication from pBR322 is indicated. Figure 6.
  • Scan of the acpP mRNA for target sites susceptible to inhibition by antisense peptide-PNAs The 5' region of the E coli acpP mRNA is shown, representing the X-axis.
  • the binding site of the antisense PNAs is shown as lines below the mRNA sequence, along with the name corresponding to the peptide-PNA.
  • the lines within the graph indicate the minimum inhibitory concentrations (MIC) determined for each peptide-PNA as indicated on the Y-axis. Peptide-PNAs found to not be inhibitory at the highest concentration tested are indicated at the top.
  • the green fluorescence expression plasmid pSPT-Pxyl-GFP The insert shown at the top indicates the cloning strategy, construction and peptide-PNA 1900 binding site.
  • the arrows indicate antibiotic resistance markers, the xylose promoter and the origin or replication from pBR322 is indicated.
  • B. subtilis cells Green fluorescence protein expression and antisense inhibition in B. subtilis 168. After overnight growth in the presence or absence of PNA, B. subtilis cells were visualised by light microscopy at 100 x magnification. The top panels illustrate control cells that did not carry the gfp reported gene. Cells expressing gfp were treated with PNA as indicated. Figure 9.
  • LacZ expression and inhibition in E. coli with a range of differently sized PNAs LacZ expression and inhibition in E. coli with a range of differently sized PNAs.
  • the histograms at the left illustrate results for wild type strain K12 and those at the right are for the permeable mutant strain AS19. Values represent LacZ activities relative to 10 control levels for cultures grown in 10% LB broth.
  • the PNAs used were 12m: PNA1878; 15: PNA1432; 14: PNA1835; 12: PNA1834; 11: PNA1877; 10: PNA1833; 9: PNA1876; 8: PNA1832; 7: PNA1875.
  • LacZ expression and inhibition in E. coli with PNA, free peptide and a peptide-PNA 15 conjugate The values represent relative enzyme activities in E. coli K12 cultures grown in 10% LB broth.
  • Antisense effects with 12 mer PNA 1834 open circles
  • free peptide KFFKFFKFFK SEQ ID NO: 1
  • a mixture of free PNA and peptide closed squares
  • the peptide-PNA conjugate 1900 closed circles.
  • A E coli K12 cells.
  • B E. coli K12 cells with plasmid pKK223-3/acpP-i . Cultures were grown in the absence (closed circles) or presence (open squares) of added PNA (2 ⁇ M). The structure of the wild type chromosomal and mutant
  • HeLa cell culture noninvasive E. coli infection and antimicrobial PNA treatment.
  • the images show HeLa cell cultures grown in MEM , 10% FCS.
  • the column at the left 35 shows HeLa cell cultures without added E coli
  • the center column shows HeLa cell cultures that were inoculated with E. coli K12
  • the right column shows HeLa cell cultures that were inoculated with E coli K12/acpP-i.
  • the top panels of each column show cultures not treated with PNA, and the rows below show cultures treated with increasing amounts of anti-acpP PNA.
  • the colony forming units determined 24 hrs post inoculation are indicated.
  • B. subtilis growth and inhibition by an anti-acpA peptidePNA Cell growth is indicated by increased turbidity at 550 nm. Culture were grown in the absence (closed circles) or presence of different concentration the anti-acp-4 PNA (open triangles with peak upwards, 0.5 ⁇ M; closed diamonds, l ⁇ M; hatched squares, 2 ⁇ M; closed triangles, 3 ⁇ M; open triangles with peak downwards, 4 ⁇ M; open squares, 5 ⁇ M).
  • CFU colony forming units
  • E. coli strain AS19 (Nikaido, 1994) was obtained from S. Pedersen (Univ. Copenhagen), E. coli strain DH5 ⁇ was from Life Technologies (Gaithersburg, USA) and wild type E. coli strain K12 was from the E coli Genetic Stock Center (Yale University).
  • peptide-PNA conjugates were synthesized by conventional maleimide coupling chemistry as will be reported in detail elsewhere. Briefly, PNA (1 mg, 0.29 ⁇ mol (1 equivalent)) was dissolved in minimal volume (typically 2 ml) of NMP: DMSO (8:2, v/v). SMCC maleimide reagent (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; Pierce) (0.5 mg, 5 equivalents) and DIEA (40 ⁇ L) were added to PNA solutions and this was shaken gently at room temperature for 2.5 hr.
  • the PNA-maleimide conjugates were precipitated by addition of excess dry cold ether (10 ml) and washed with ether containing 10% NMP (5 x 10 ml) in order to remove excess SMCC.
  • the PNA-maleimide conjugate was dried under nitrogen, dissolved in 200 ⁇ L of DMF and allowed to react with the cysteine reduced peptide (3.25 mg, 5 equivalents) overnight.
  • the conjugates were purified by HPLC (C 18 column) using acetonitrile-H 2 0 containing 0.1% TFA as eluent and further characteized by MALDI-TOF mass spectrometry.
  • E. coli cultures were initiated with 10 4 cfu added to 100 ⁇ l of 10% LB broth in 1.5 ml siliconized microdilution tubes.
  • the lacZ gene was induced with 50 ⁇ M isopropyl ⁇ -D- thiogalactopyranoside (IPTG). After overnight growth at 37°C, LacZ activity was assayed as described (Miller, 1972). Luciferase gene expression and inhibition was assayed using E. coll K12 carrying pBestluc and the Luciferase Assay System (Promega, cat no E1500).
  • E coli K12 cells were inoculated at 10 5 cfu/ml in 100 ⁇ l of MH broth in a low binding 96 well microtiter plate (Costar 7424) with PNAs included. Plates were incubated at 37°C in a Molecular Dynamics Spectromax spectrophotometer set to shake the plate for 5 seconds and record turbidity at 550 nm at 5-minute intervals.
  • the minimum inhibitory concentrations (MICs) for PNAs targeted to the particular binding sites were determined by monitoring the growth of E. coli K12 in the presence of a range of PNA concentrations. The lowest concentration that prevented growth by visual inspection after overnight (16h - 24h as indicated) incubation from an inoculum of 10 s cfu/ml was taken as the MIC.
  • the growth media varied from experiment to experiment. Most MIC experiments were performed in LB broth diluted to 10% of normal strength; however, a few experiments were performed in full strength Mueller Hinton (MH) broth or 5 Minimal Essential Media alpha supplemented with 10% fetal calf serum as indicated. The highest concentrations of PNA tested varied from experiment to experiment and are indicated.
  • E. coli cells in logarithmic growth were diluted to 10 5 cfu/ml and incubated with added PNA 10 at 37°C in Costar 7424 microtitre plate wells. Samples were taken at time intervals, plated onto LB agar and grown at 37°C overnight and counted.
  • Electrocompetent DH5 ⁇ were prepared as described (Sambrook et al. 1989). The electrocompetent DH5 ⁇ (35 ⁇ l) were transformed with 1 ⁇ l of pKK223-3/acpP-l (30 ng/ml) 15 using a BioRad GenePulser II, Hercules, CA, USA. The sample was electroporated in a 1 mm gap width cuvette at 1.8 kV, 25 mF, 200 ohms for 2.5 ms and the cells were left to recover in 250 ⁇ l of SOC-media at 37°C for 40 minutes.
  • the bacteria were diluted to a 20 concentration of 1.25 x 10 5 cells/ml in 10% LB low salt. Aliquots of 80 ⁇ l ( ⁇ 10 4 cells) were added to the wells of an "Ultra low attachment 96 well plate with lid", along with 20 ⁇ l of PNA SP4 (1.0 ⁇ M final concentration). The plate was incubated under the same conditions as described above.
  • Electrocompetent DH5 ⁇ were prepared as described (Sambrook et al. 1989) and transformed with 1 ⁇ l of pKK223-3/acpP-J (30 ng/ml) and the cells were left to recover in 250 ⁇ l of "SOC-media at 37°C for 40 minutes, as described (Sambrook et al. 1989).
  • the bacteria were diluted to a concentration of 1.25 x 10 5 cells/ml in 10% LB low salt (5 g of NaCl/l). Aliquots of 80 ⁇ l ( ⁇ 10 4 cells) were added to the wells of an "Ultra low attachment
  • Antisense PNAs are larger than most drugs and PNA size or length is likely to be an important parameter for efficiency.
  • PNAs targeted to the start codon region of the chromosomal ⁇ - galactosidase gene (lacZ) (see Figure 9 and Table 5) were synthesized over the 7 to 15 mer size range as described in the Materials and Methods section.
  • the E coli outer cell wall is a major barrier to PNA (Good 2000). Recently the synthetic peptide (KFFKFFKFFK (SEQ ID NO: 1)) was shown by Vaara (1996) to improve the permeability. To test whether this cell wail permeating peptide, when conjugated to PNA oligomers, could enhance the uptake and thus efficacy of antisense PNAs, the 12 and 15 mer anti-/acZ PNAs were synthesized with the KFFKFFKFFK (SEQ ID NO: 1) peptide attached via a flexible ethylene glycol-lysine linker (egl) (AEEA-OH spacer Cat# GEN063030 AppliedBiosystems, Framingham, MA, USA) as described in the Materials and Methods section and in Table 5.
  • a flexible ethylene glycol-lysine linker egl
  • peptide-PNA conjugates were assembled via maleimide SMCC coupling (PNA 1978) and the ethylene glycol linker (PNA 1900). As seen in Table 5, a peptide-PNA conjugate with the SMCC linker is more than 10 fold less potent than one with the ethylene glycol linked PNA (PNA 1900).
  • peptides other than the KFFKFFKFFK (SEQ ID NO: 1) motif are able to carry PNA oligomers into bacteria as indicated by the comparable potencies for PNAs 1978, p29, p30, p31 and p32 (Table 5).
  • Example 3 Peptide-PNA conjugate inhibition of bacterial growth.
  • MICs Minimum inhibitory concentrations (MICs) of the resulting peptide-PNA conjugate (PNA1934) as well as 4 "non-conjugated" versions (PNA 1143, 1410, 1406 and 1407; see Table 5) were found by establishing the lowest PNA concentrations that prevented E. coli growth by visual inspection after overnight (16h) growth from an inoculum of 10 5 cfu/ml in 10% LB broth.
  • the MIC of PNA1934 was also established in Mueller Hinton broth (Cat* M9677, Sigma Chemical Co. St. Louis, MO. USA).
  • the maximum concentrations of anti- ⁇ - sarcin related PNAs were 5 ⁇ M.
  • the peptide-PNA conjugate (PNA1934) exhibited an MIC of 3 ⁇ M against E. coli K12 grown in Mueller Hinton broth and an MIC of 700 nM when grown in diluted LB broth (Table 5). Free peptide and PNA 1143 were not inhibitory at these concentrations, showing that conjugation of the peptide with PNA is needed for potent growth inhibitory effects of "non-permeable" E. coli.
  • Example 4 Peptide-PNA conjugate targeted against the essential acpP gene.
  • E. coli K12 was pre-grown overnight in 2 ml of 10% LB low salt media (Cat#L1703, Saveen, Malm ⁇ , SE (note, this is not standard LB) at 37°C with constant shaking at 225 rpm. Optical density was measured at 550 nm the following day and the culture was diluted to 1 xlO 5 bacteria per ml in Mueller Hinton broth (Cat#L1703, Saveen, Malm ⁇ , SE). Aliquots (80 ⁇ l) corresponding to approximately 10 4 cells were added to the wells of an "Ultra low attachment 96 well plate with lid" (COSTAR ® , Corning Inc., NY).
  • peptide-PNA constructs were diluted in water and aliquots were transferred into microtitre plate wells to provide a PNA concentration ranging from 100 to 1000 nM and a final culture volume of 100 ⁇ l.
  • the 96 well plate was incubated for 24 hours in a VERSAmax spectrophotometer (Molecular Devices Corporation, CA, USA) at 37°C with shaking and OD measurements each five minutes. Shaking time was set to five seconds and optical density was measured at 550 nm.
  • the minimum inhibitory concentrations (MICs) for peptide-PNAs targeted to different acpP mRNA binding sites were determined by monitoring the growth of E coli K12 in the presence of a range of peptide-PNA concentrations. The lowest concentration that prevented growth after 24 hours of incubation was taken as the MIC. The highest concentrations tested were 10 ⁇ M of the test substance.
  • the peptide-PNA binding sites and the resulting MICs are illustrated in Figure 6.
  • the results indicate that the start codon region including the Shine Delgarno sequence is particularly susceptible to antisense inhibition. All but one of the peptide-PNAs targeting this region showed low MIC values, whereas peptide-PNAs targeting downstream sites were not inhibitory at the highest concentrations tested (10 ⁇ M).
  • the results support the idea that the start codon region is a susceptible target sequence for antisense inhibition by peptide-PNAs. Also, the results suggest that the number of susceptible binding sites within mRNA is restricted and this improves the probabilities for gene specific effects.
  • the SP4 PNA complementary to the start codon region of the essential acpP gene was tested for its bacteriocidal effects and its specificity.
  • the bacteriostatic and bacteriocidal effects of the anti-acpP peptide-PNA were demonstrated in two ways.
  • the MICs of SP4 and a control peptide-PNA with a scrambled PNA sequence were determined as described in the Materials and Methods section.
  • the control peptide-PNA had a 15 fold higher MIC than the fully matched version SP4 (Table 5). This result is consistent with an acpP specific inhibitory effect for SP4.
  • the E coli acpP gene was cloned and selected bases altered to eliminate the potential for PNA binding, but still encode native Acp as described.
  • Primers complementary to each end of the acpP gene were designed and ordered from Interactiva Biotechnologie (Ulm, Germany).
  • the E coli acpP gene was amplified by PCR using the primers, 5 '-GAGAATTCATGAGCACTATCGAAGAAC-3 ' (SEQ ID NO: 15) and 5 '- AGTTAAGCTTGACCGCCTGGAGATGTT -3 ' (SEQ ID NO: 16).
  • These primers contain sites for the restriction enzymes EcoR I and Hind III and are designed to mutagenise the bases between the Shine-Delgarno sequence and the AUG initiation codon (see Figure 2, 6 and 14).
  • a primer extension reaction was performed with 10 pmol of each of the primers, 0.2 mM dNTP mix, 2.5 U Taq polymerase (Amersham Pharmacia) and 1/40 of a "normal sized" colony of K12 cells (grown overnight on an LB plate at 37°C) in a total volume of 100 ⁇ l buffered with PCR-buffer (the above products were included in "Advantage ® PCR Kit", Clonetech, USA).
  • the reaction was incubated in a "Peltier Termal Cycler" (PTC-225, MJ Research, NY) with 1 minute at 94°C followed by 1 minute at 50°C and 1 minute at 70°C for 30 cycles.
  • the amplified sample was run on a 1.5% agarose gel for 1 hour. The gel was stained with ethidium bromide and the bands were visualised with long UV light. A fragment corresponding in size to the acpP gene (275 nt) was extracted and cleaved with 0.5 ⁇ l each of the enzymes EcoR I and Hind III (New England Biolabs, USA), and ligated into the expression vector pKK223-3 (Amersham-Pharmacia, Uppsala, SE). A map of the resulting construct is shown in Figure 5.
  • Example 6 Selection of acpP-1 containing transformed cells in a mixed culture.
  • E. coli K12 and K12/pKK223-3/acpP-i were pre-grown overnight in 2 ml of 10% LB low salt media at 37°C with constant shaking at 225 rpm.
  • Optical density was measured at 550 nm the following day and the cultures were diluted to 2.5 x 10 s bacteria per ml in 10% Luria Bertani broth. Aliquots (40 ⁇ l) corresponding to approximately 10 4 cells of mono or mixed cell types were added to the wells of an "Ultra low attachment 96 well plate with lid" (COSTAR ® , Corning Inc., NY).
  • PNA treated samples included 20 ⁇ l of PNA SP4 (1.0 ⁇ M final concentration), and 40 ⁇ l of 10% LB low salt were added where needed to make a total culture volume of 100 ⁇ l.
  • the 96 well plate was incubated for 24 hours in a VERSAmax spectrophotometer (Molecular Devices Corporation, CA, USA) at 37°C with shaking and OD measurements each five minutes. Shaking time was set to five seconds and optical density was measured at 550 nm.
  • Samples of 2 ⁇ l were harvested from the mono, mixed and transformed cell cultures after 0, 1, 2, 3 and 24 hours.
  • the harvested samples were mixed with 98 ⁇ l of water, and 50 ⁇ l of each mixture were plated out on standard Luria-Bertani plates (Sambrook et al. 1989).
  • the remaining diluted cultures were used to make further dilutions that were also spread out on LB-plates.
  • the plates were incubated at 37°C for 48 hours.
  • colonies derived from each harvest time as many as possible and over one hundred were picked and transferred individually to LB-plates containing ampicillin (75 ⁇ g/ml).
  • the LBA- plates were incubated at 37°C for 24 hours and the number of growing colonies was counted relative to the total number of colonies transferred.
  • the table shows the growth of wild type and pKK223-3/acpP-i carrying (K12/acpP-I) E coli K12 on LB plates.
  • the cells were pre-grown for different times as mono or mixed cultures in the presence or absence of 1 ⁇ M PNA SP4, as indicated.
  • pKK223-3/acpP-I not only carries a PNA SP4 target site altered copy of the acpP gene, but also the ampicillin resistance gene, the amount of plasmid carrying colonies in the mixed culture was deduced by colony transfer to LB-Amp plates. The amount of Amp r clones is seen in the right-most column.
  • antisense PNA can be used to select for specific strains from a mixed E. coli culture when targeted against an essential gene.
  • Example 7 Selection of acpP-1 transformants following electroporation.
  • Electrocompetent DH5 ⁇ were prepared and transformed with 1 ⁇ l of pKK223-3/acpP-i (30 ng/ml) as described in the Materials and Methods section.
  • the bacteria were diluted to a concentration of 1.25 x 10 5 cells/ml in 10% LB low salt (5 g NaCI/l). Aliquots of 80 ⁇ l ( ⁇ 10 4 cells) were added to the wells of an "Ultra low attachment 96 well plate" (Costar), along with 20 ⁇ l of PNA SP4 (1.0 ⁇ M final concentration) and incubated in a VERSAmax spectrophotometer (Molecular Devices Corporation, CA, USA) at 37°C with shaking and OD measurements each five minutes. Shaking time was set to five seconds and optical density was measured at 550 nm.
  • VERSAmax spectrophotometer Molecular Devices Corporation, CA, USA
  • Samples of 2 ⁇ l were harvested from the electrotransformed cell cultures after 0, 2, 4, 6, 18, and 24 hours.
  • the harvested samples were mixed with 98 ⁇ l of water, and 50 ⁇ l of each mixture were plated out on standard Luria-Bertani plates (Sambrook et al. 1989).
  • the remaining diluted cultures were used to make further dilutions that were also spread out on LB-plates.
  • the plates were incubated at 37°C for 48 hours.
  • colonies derived from each harvest time as many as possible and over one hundred were picked and transferred individually to LB-plates containing ampicillin (75 ⁇ g/ml).
  • the LBA- plates were incubated at 37°C for 24 hours and the number of growing colonies was counted relative to the total number of colonies transferred.
  • the table shows the number of colony forming units (cfu) in a culture of DH5 ⁇ cells transformed with pKK223-3/acpP-l and grown in the presence of 1 ⁇ M PNA SP4 for different times.
  • the right-hand column shows the amount of Amp r cells.
  • Plasmids were purified using GFX Micro Plasmid-prep Kit (Amersham Pharmacia, Uppsala, SE) fractionated in a 1% agarose/Tris-borate-acetate gel electrophoresis, stained with ethidium bromide (0.5 ⁇ g/ml) and photographed.
  • B. subtilis wild type strain 168 was obtained from the Institute Pasteur strain collection, France. The PNA was synthesised as described in the Materials and Methods section. Luria- Bertani medium was used for growing and manipulating E. coli and B. subtilis. Cultures were grown at 37°C on a rotary shaker (222 r.p.m.) overnight. Antibiotics were used at the following concentrations: tetracycline, 10 ⁇ g/ml; ampicillin 75 ⁇ g/ml.
  • Plasmids used are listed in Table 4. All plasmid constructs were first propagated in E coli DH5 ⁇ and then transferred to B. subtilis 168. Competent E.coli cells were prepared and transformed by the method of Sambrook et al. (1982). B. subtilis was transformed by electroporation as described below. E coli transformants were selected on LB (Difco, LePont de Claix, France) plates containing 75 ⁇ g of ampicillin ml "1 and B. subtilis transformants on LB agar plates containing 10 ⁇ g of tetracycline ml "1 . To optimize expression of gfp in gram positive bacteria we introduced a consensus Shine-Delgarno sequence GGAGG just upstream of the start codon.
  • the plasmid pEGFP was used as template; this plasmid contains a gfp derivative with enhanced fluorescence properties (Clonetech, Palo Alto, CA). Also, to enable comparison with previous antisense PNA experiments with E. coli, the target site for peptide-PNA 1900 was introduced at the start codon region.
  • the gpf gene was amplified with T. aquaticus DNA polymerase (Amersham Pharmacia), using a PCR recaction consisting of 95°C for 30 sec, 55°C for 1 min and 72°C for 1 min which was repeated 30 times.
  • the fragment was first cut with Sacl and analysed in 1% agarose gel.
  • the cleaved ⁇ 800-bp PCR fragment was gel purified and ligated to the Sacl/Smal site of the polylinker region of the E. coli gram positive shuttle vector pSPT245.
  • the recombinant plasmid containing the promoterless gfp reporter gene, designated pSPT245-GFP was amplified in E. coli and verified by restriction analysis.
  • Plasmid DNA was extracted by using the GFX Micro Plasmid Preo Kit (Amersham Pharmacia Biotech Inc,USA). Plasmid pSPT245-GFP+-Pxyl, carrying the gfp gene under the control of the xylose promoter, was constructed by cloning a Sphl/BamHI fragment containing the entire xylR gene and xylA promoter region from pASl into pSPT245-GFP cut with Sp ⁇ I and Bam l.
  • the electrocompetent cells were mixed with an equal volume of 0.5 M sucrose and incubated 5 min at room temperature.
  • 50 ⁇ l of cell suspension was incubated with 1.5 ⁇ l of DNA for 30 min at room temperature.
  • the sample was transferred to a 1 mm electroporation cuvette and pulsed once at 2 kV with a 25 ⁇ F capacitor (10 kV/cm field strength for 2.5 ms) in a BioRad GenePulser II, Hercules, CA, USA.
  • the sample was immediately resuspended in 950 ⁇ l of TSB broth and transferred to a 2.5 ml tube and shaken for 1.5 h at 225 rpm at 37°C.
  • the cells were then plated on LB agar plates containing the appropriate selective agent and incubated at 37°C for 24-48h.
  • B. subtilis growth and antisense PNA treatment B. subtilis cells were pre-grown overnight in 2 ml of Mueller Hinton broth at 37°C with constant shaking at 225 rpm, with 5 mg/ml tetracyline. Optical density was measured at 550 nm the following day and the cultures were diluted to 2.5 x 10 5 bacteria per ml in 10% Luria Bertani broth. Aliquots (40 ⁇ l) corresponding to approximately 10 4 cells of mono or mixed cell types were added to the wells of an "Ultra low attachment 96 well plate with lid" (COSTAR ® , Corning Inc., NY).
  • PNA treated samples included 20 ⁇ l of PNA SP4 (1.0 ⁇ M final concentration), and 40 ⁇ l of Mueller Hinton broth were added where needed to make a total culture volume of 100 ⁇ l.
  • the 96 well plate was incubated for 24 hours in a VERSAmax spectrophotometer
  • B. subtilis cells were collected by centrifugation at 5000 rpm for 5 min. The cell pellets were resuspended in 1 X PBS buffer and fluorescence microscopy was performed using a Leica DMRXA microscope with a cooled frame CCD camera using excitation and emission wavelengths of 488 and 507 nm for GFP and 490 and 520 nm for FITC, with OpenlabTM image analysis and presentation.
  • the expression vector was constructed to provide a binding-site for the peptide-PNA 1900 (Figure 7). This PNA was shown previously to provide potent antisense inhibition in E. coli (Good et al. 2001).
  • the transformed version of B. subtilis showed strong green fluorescence when visualised by fluorescence microscopy and the parent strain showed very low background fluorescence ( Figure 8).
  • the cells grew at a normal rate and appeared normal when viewed under standard white light conditions. When viewed using fluorescence microscopy, the visible green fluorescence was markedly or entirely reduced by the peptide-PNA 1900 ( Figure 8).
  • a control peptide-PNA (1901) did not reduce visible green fluorescence.
  • antisense peptide-PNA entered B. subtilis cells and inhibited gfp expression by binding to gfp mRNA.
  • antisense peptide-PNA inhibiton strategies can be extended to B. subtilis and possibly other gram positive bacteria.
  • Example 9 Using antisense PNA directed against the acpP gene to remove bacterial growth from a cell culture of human HeLa cells.
  • HeLa cells were obtained from CloneTech, Palo Alto, CA, USA, Cat#C3001-l. HeLa cells were cultured in Minimal Essential Medium alpha (MEM ⁇ ), 10% fetal calf serum (Life Technologies). Prior to infection, HeLa cells were suspended by scraping, transferred to 96 well Falcon 3872 plates and grown to approximately 30% confluence. Freshly grown E. coli K12 cells were added at 10 5 cfu/ml. Following infection, PNA solution or an equivalent volume of water was added and the plates were incubated overnight at 37°C in 5% C0 2 .
  • MEM ⁇ Minimal Essential Medium alpha
  • E. coli K12 cells were added at 10 5 cfu/ml. Following infection, PNA solution or an equivalent volume of water was added and the plates were incubated overnight at 37°C in 5% C0 2 .
  • the PNA did not visibly affect HeLa cell growth at the highest concentration tested (20 ⁇ M) ( Figure 14), and PNA at 2 ⁇ M or higher appeared to fully cure the HeLa culture of the infection. No live bacteria were detected from these samples in an agar colony forming unit test, where dilutions were spread on LB plates, incubated overnight at 37°C and the number of colony forming unit detected was used to calculate the value for cfus/ml in the sample, confirming that the anti-acpP PNA is bactericidal under these conditions. Again, the sequence specificity of the effect was evaluated in cultures infected with E. coli carrying the binding-site-altered acpP allele (K12-acpP-l) and this infection was resistant to PNA up to at least 20 ⁇ M ( Figure 14).
  • Example 10 Bactericidal antisense PNA effects against B. subtilis.
  • Bacterial strain and PNA Bacillus subtilis wild type strain 168 was obtained from the Institute Pasteur strain collection, France. The PNA was synthesised as described in the Materials and Methods section.
  • Cultures were pre-grown overnight in 2 ml 50% Mueller-Hinton broth at 37°C with constant shaking at 225 rpm. Optical density was measured at 550 nm the following day and the cultures were diluted to 2.5xl0 4"5 bacteria per ml. Aliquots (40 ⁇ l) corresponding to approximately 10 4 cells of mono or mixed cell types were added to the wells of an "Ultra low attachment 96 well plate with lid” (COSTAR ® , Corning Inc., NY). PNA treated samples included 20 ⁇ l PNA (1.0 ⁇ M final concentration), and medium was added where needed to make a total culture volume of 100 ⁇ l.
  • the 96 well plates were incubated in a VERSAmax spectrophotometer (Molecular Devices Corporation, CA, USA) at 37°C with shaking and OD measurements each three minutes. Shaking time was set to fifteen seconds and optical density was measured at 550 nm.
  • VERSAmax spectrophotometer Molecular Devices Corporation, CA, USA
  • Viable cell counts B. subtilis grown to logarithmic growth were diluted to 10 6 CFU/ml and incubated with added PNA. Samples were taken at time intervals, plated onto LB agar and grown at 37°C overnight.
  • the antibacterial effects of the anti-acp ⁇ peptide-PNA conjugate on B. subtilis cells were further examined by assessing cell viability.
  • CFU colony-forming unit
  • PNA designation and sequence bases ( ⁇ M) ( ⁇ M) ( ⁇ M) anti-/ac2 PNAs.
  • MIC Minimal inhibitor concentrations were the lowest PNA concentrations that prevented E coli growth by visual inspection after overnight (16h) growth from an inoculum of 10 5 cfu/ml.
  • the J bases indicate pseudo-isocytosine.
  • smcc designates PNA-maleimide - cystein-peptide coupling via the smcc maleimide-active ester reagent. Cells were grown in LB broth diluted to 10% of normal strength, except ! where indicated as growth in ( ) full strength Mueller Hinton (MH) broth or (c) Minimal Essential Media alpha, 10% fetal calf serum.
  • PNA peptide nucleic acid
  • 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. Proc. Natl. Acad. Sci. U S A 97, 418-423 (2000).
  • PNA Peptide nucleic acid
  • Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382-388 (1994).

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Abstract

L'invention concerne un nouveau procédé permettant de sélectionner des cellules génétiquement modifiées sans antibiotiques. On a découvert que des molécules antisens ciblées vers un gène cellulaire essentiel inhibe la croissance et sont appropriées comme agent de sélection de croissance de cellules transformées à l'aide d'un plasmide portant une version altérée dudit gène essentiel. Les résultats obtenus montrent qu'on peut utiliser des molécules antisens pour sélectionner des microbes transformés désirés contre un gène microbien essentiel sans antibiotique. Cette technologie est utilisée en génie génétique pour la recherche de croissance et d'isolation d'organismes transformés, et pour la maintenance de la croissance industrielle de ces organismes transformés, par exemple, dans la production de protéines mises au point par génie génétique utilisée comme alternative environnementale plus sure par rapport aux méthodes des sélection traditionnelles à base d'antibiotiques.
PCT/DK2002/000208 2001-03-29 2002-03-26 Selection de souche bacterienne sans antibiotique a l'aide de molecules antisens Ceased WO2002079467A2 (fr)

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CN114958896B (zh) * 2021-11-12 2023-06-27 淮阴师范学院 一种提高乳酸片球菌的电转化效率的方法
WO2023093960A1 (fr) * 2021-11-26 2023-06-01 Syddansk Universitet Conjugué peptide-oligonucléotide modifié

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