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WO1998003533A1 - Oligonucleotides anti-sens utilises comme agents antibacteriens - Google Patents

Oligonucleotides anti-sens utilises comme agents antibacteriens Download PDF

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Publication number
WO1998003533A1
WO1998003533A1 PCT/US1997/012961 US9712961W WO9803533A1 WO 1998003533 A1 WO1998003533 A1 WO 1998003533A1 US 9712961 W US9712961 W US 9712961W WO 9803533 A1 WO9803533 A1 WO 9803533A1
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oligonucleotides
bacterium
oligonucleotide
composition
genus
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PCT/US1997/012961
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WO1998003533B1 (fr
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Amy Arrow
Roderic M. K. Dale
Theresa L. Thompson
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Oligos Etc. And Oligos Therapeutics, Inc.
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Priority to AU38118/97A priority Critical patent/AU3811897A/en
Publication of WO1998003533A1 publication Critical patent/WO1998003533A1/fr
Publication of WO1998003533B1 publication Critical patent/WO1998003533B1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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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
    • C12N15/11—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/311—Phosphotriesters
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/312—Phosphonates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/313—Phosphorodithioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/314—Phosphoramidates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/315—Phosphorothioates
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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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to methods for treating an animal, including a human, having a bacterial infection which comprise administering an oligonucleotide specifically targeted to, or otherwise capable of interacting with, a bacterial sequence, or nucleic acid binding protein.
  • the antibacterial oligonucleotide inhibits the growth of the bacteria, blocks the expression of virulence factors or genes involved in the transfer of genetic information, or kills the bacteria.
  • the oligonucleotide may also be targeted to an antibiotic resistance gene in order to render the bacteria sensitive to an otherwise ineffective antibiotic.
  • the invention also relates to nuclease resistant oligonucleotides that are effective in inhibiting the growth of, or killing, pathogenic bacteria.
  • Streptococcus pneumoniae is a leading cause of morbidity and mortality in the United States (M.M.W. ., Feb. 16, 1996, Vol. 45, No. RR-1) . Each year these bacteria cause 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 7,000,000 cases of otitis media. Case fatality rates are greater than 40% for bacteremia and greater than 55% for meningitis, despite antibiotic therapy. In the past, Streptococcus pneumoniae were uniformly susceptible to antibiotics; however, antibiotic resistant strains have emerged and are becoming widespread in some communities.
  • Antisense polynucleotides are useful for specifically inhibiting unwanted gene expression m mammalian cells. They can be used to hybridize to and inhibit the function of an RNA, typically a messenger RNA, by activating RNase H or physically blocking the binding of ribosomes or proteins, thus preventing translation of the mRNA. Antisense oligonucleotides also include RNAs with catalytic activity (nbozymes) , which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mediating a cleavage reaction.
  • RNAs with catalytic activity RNAs with catalytic activity
  • Antisense oligonucleotides that bind to the DNA at the correct location can also prevent the DNA from being transcribed into RNA. These antigene oligonucleotides are believed to bind to double-stranded DNA (forming triple- stranded DNA) and thereby inhibit gene expression.
  • antisense oligonucleotides have emerged as a powerful new approach for the treatment of certain diseases.
  • the preponderance of the work to date has focused on the use of antisense oligonucleotides as antiviral agents or as anticancer agents (Wickstrom, E., Ed., Prospects for Antisense Nucleic Ac d Therapy of Cancer and AIDS, New York: Wiley-Liss, 1991; Crooke, S.T. and Lebleu, B., Eds.,
  • antisense oligonucleotides as antiviral agents.
  • Agrawal et al . report phosphoramidate and phosphorothioate oligonucleotides as antisense inhibitors of HIV (Agrawal et al . , Proc. Natl. Acad . Sci . USA 8jj: 7079-7083 (1988)).
  • Zamecnik et al disclose antisense oligonucleotides as inhibitors of Rous sarcoma virus replication in chicken fibroblasts (Zamecnik et al . , Proc . Natl. Acad. Sci . USA £:4143-4146 ( 1986 ) .
  • oligonucleotides should be advantageous as an approach to treating bacterial infectior, because sequences can be specifically designed to inhibit bacterial growth while not interfering with the metabolism of mammalian cells.
  • oligonucleotides have been shown to nonspecifically stimulate the immune system (Yamamoto et al . , Antisense Res. Devel. 4 . : 119-122 (1994); Krieg et al . , Nature 374 : 546-549 (1995)). Since current antibiotics generally function by arresting bacterial growth until the immune system can respond to the infection (Myrvik, Fundamentals of Medical Bacteriology, 1974, Lea & Febiger, Publishers), the use of oligonucleotides as antibiotics may provide both a nonspecific stimulation of the immune system as well as the relatively specific inhibition of the growth of a particular bacteria.
  • antisense oligonucleotides should be ideally suited to the treatment of bacterial infections involving the liver and spleen as well as systemic bacteremia and septicemia.
  • reports of antisense oligonucleotide- mediated gene inhibition in bacteria have attempted to circumvent the perceived problem of the rigid cell wall by conducting experiments in cell -wall deficient strains (Jayaraman et al . , Proc . Natl.
  • Lupski et al . Pat. No. 5,294,533 ('533 patent), stated that antisense oligonucleotides can preferentially inhibit the growth of Gram negative and Gram positive bacteria in a mixed culture of Gram negative and Gram positive bacteria.
  • Lupski et al . also taught that end-capped oligonucleotides should be used (see column 4, lines 39-42), but since end- capping does not provide protection from intracellular endonucleases (see the discussion of Hoke et al . above), one skilled in the art would not expect the method of Lupski et al . to work.
  • the '533 patent does not provide an enabling description of the use of antisense oligonucleotides to inhibit the growth of bacteria in vi vo in mammals.
  • the '533 patent did not disclose the genotype of the bacteria used in the study. Thus, there is no way to establish whether clinical isolates were used or permeability enhanced bacterial mutants were used. Additionally, the '533 patent does not provide adequate teaching to allow one to discern whether or not the described bacteria had been previously rendered competent by established prior art methods. In view of this lack of disclosure, the '533 patent does not teach methods that are broadly applicable to clinically significant bacterial infections in mammals. The prior art teaches the inherent difficulty of successfully using oligonucleotides to inhibit the growth of intact bacteria (Jayaraman et al . and Ciferri et al . ) , and the '533 patent does not provide sufficient disclosure to refute the clear teaching in the prior art. Instead, the
  • oligonucleotides are not able to inhibit the growth of unmodified (intact) bacteria. Additionally, the last reference (Lupski) provides no teaching of how to inhibit the growth of intact bacteria, and provides no illustrative examples that such inhibition is indeed possible.
  • oligonucleotides may affect antibacterial efficacy. These features include, but are not limited to: 1) the dose of oligonucleotide; 2) the length of the oligonucleotide; 3) the growth conditions used during the in vi tro assay; 4) the chemical backbone of the oligonucleotide; and 5) the method of post-synthesis purification. Each of these features are discussed in greater detail below.
  • oligonucleotide may significantly effect the observed amount of growth inhibition.
  • Fig. 1 shows that the percent of inhibition varies from 100% down to about 19% as the dose of oligonucleotide is reduced from 285 ⁇ M to 5 ⁇ M in a standard MIC assay (described in Section 4.5, infra) .
  • a standard MIC assay described in Section 4.5, infra
  • the length of the oligonucleotide is directly related to its ability to specifically bind and inhibit the normal function of the target sequence. Shorter oligonucleotide sequences generally have a reduced Tm (duplex melting temperature) and are thus more likely to cause undesirable side effects of nonspecific binding or have no effect.
  • Tm duplex melting temperature
  • Gao et al . , Molec . Pharm . 41 : 223- 229 (1992) have shown that, using an in vi tro enzymatic assay, the inhibitory effect of an oligonucleotide sequence increased as the length of the oligonucleotide was progressively increased from a 7mer up to a 28mer. Gao et al .
  • oligonucleotides that were a maximum of only 12 bases in length. Typically, oligonucleotides as short as the disclosed 12mers show a high degree of nonspecific binding. Lupski chose sequences of about 25 bases in length but the majority of the disclosed sequences comprised a high degree of degeneracy which allows for binding to multiple target sites.
  • oligonucleotides comprising bases such as mosine, or "N" (which indicates the use of A, C, G, or T) , are usually produced when one wishes to allow binding to sequences where the precise target sequence is unknown (Ohtsuka et al . , J. Biol . Chem. 260 :2605 (1985)). Sequences with such broad based homology run the risk of nonspecific binding to host sequences and associated toxicity effects. Additionally, Lupski 's teaching is inherently suspect given that no data demonstrating the inhibition of bacterial growth was provided.
  • oligonucleotide sequences generally have reduced T 's.
  • the oligonucleotides taught by Rahman, Jayaraman, Gasparro, and Chrisey were generally so short that the Tm's for the oligonucleotide- target sequence hybrids were usually below 37° C.
  • the 12mer phosphorothioate sequence taught by Chrisey has a predicted Tm of 28.9° C
  • the 9mer taught by Gasparro had a predicted Tm of 24.7° C
  • oligonucleotides of the length taught by these references are generally not useful as antisense or antigene agents under physiologic conditions.
  • the growth rate and conditions under which antibiotic susceptibility are measured may profoundly effect a bacterium's sensitivity to antibacterial agents (Arrow et al . , Antimicrob. Agents Chemother . 26 . : 507 (1984)), and the uptake of the antibiotic into the cell (Arrow et al . , Microbiol. Rev. 5_l:439-457 (1987)).
  • methods for screening oligonucleotides in vi tro for antibacterial activity should generally be conducted under standardized conditions that reflect the in vi vo circumstances of a given pathogen such as the NCCLS MIC tests (see Section 4.5, infra) .
  • a given pathogen such as the NCCLS MIC tests (see Section 4.5, infra) .
  • None of the background references recognized that growth conditions might effect the result of antibiotic susceptibility tests, and thus none of these references assayed for the inhibition of bacterial growth using the standardized growth conditions defined in the MIC tests.
  • the antibacterial efficacy of an oligonucleotide may be directly related to the relative nuclease resistance of the chemical backbone of the oligonucleotide.
  • Gasparro and Lupski did not recognize this facet of the present invention and thus did not teach oligonucleotides that were designed to be nuclease resistant. Consequently, the oligonucleotides used by Gasparro and Lupski would have been rapidly degraded by the cell (see Section 1.6, infra) , and would thus have little utility as antibacterial agents.
  • the rapid intracellular degradation of oligonucleotides is a barrier to efficient inhibition of gene expression.
  • One of the major problems in utilizing naturally occurring phosphodiester oligonucleotides is their rapid degradation by nucleases in mammalian cells or in serum-containing culture medium (Cohen, Oligodeoxynucleotides : Antisense Inhibitors of Gene Expression, Boca Raton, Fla . , CRC Press (1989)).
  • capping of oligonucleotides may provide protection from exonucleases in cell culture, the action of intracellular endonucleases is sufficient to degrade these capped oligonucleotides when they enter a cell .
  • nuclease resistant modified nucleotides include, but are not limited to, the methylphosphonates, p-ethoxy deoxyribonucleotides, p-ethoxy 2'-0-methyl ⁇ bonucleotides , 2'-0-methyl ribonucleotides , phosphorothioates, and others.
  • a brief description of representative nuclease resistant oligonucleotide backbones follows :
  • Methylphosphonate oligonucleotides in addition to exhibiting enhanced nuclease resistance, also have increased hydrophobicity over phosphodiester oligonucleotides and therefore have greater permeability to cell membranes as compared to phosphodiester or other more highly charged oligonucleotides
  • p-Ethoxy deoxyribonucleotides have an ethyl group o- Imked to the phosphate backbone
  • p-Ethoxy deoxyribonucleotides are resistant to nuclease degradation.
  • p-Ethoxy ribonucleotides have the following structure •
  • Phosphorothioates are compounds in which one of the nonbridging oxygen atoms in the phosphate backbone of the nucleotide is replaced by a sulfur atom.
  • the phosphorothioates are resistant to cleavage by nucleases and, since they have the same number of charged groups as phosphodiester oligonucleotides, have good solubility in water. These compounds also exhibit more efficient hybridization with complementary DNA sequences than the corresponding methylphosphonate analogues.
  • Methyl carbonates are compounds in which one of the nonbridging oxygen atoms in the phosphate backbone has been replaced by a methyl carbonate group.
  • 2'-0-methyl ribonucleotides are compounds in which the 2' position of the ribose sugar ring has a methoxy group in place of the normal hydroxyl group.
  • 2'-0-methyl ribonucleotides have the following general structure
  • Secondary structure can also be used to make oligonucleotides resistant to nucleases. Oligonucleotides with a hairpin loop structure extending from the 3' -terminus, stabilizing the oligonucleotide against 3 ' -nucleolytic degradation, have been reported by Khan and Coulson, Nucl . Acids Res. 21(12) :2957-2958 (1993) . The Tm of the modified oligonucleotide from its complementary mRNA target was unaffected by the presence of the loop modification.
  • end modification of oligonucleotides can also render an oligonucleotide resistant to nucleases, such as, for example, attaching cholesterol, psoralen, rhodamine, fluorescein, DNP, amine groups, biotin, inverted (3' -3' or 5' -5') linkages, and the like, to the end of the oligonucleotide in order to render it more nuclease resistant 2.0.
  • nucleases such as, for example, attaching cholesterol, psoralen, rhodamine, fluorescein, DNP, amine groups, biotin, inverted (3' -3' or 5' -5') linkages, and the like
  • the present invention relates to methods for the treatment of animals, including humans, that have a bacterial disease.
  • the preferred method of treatment comprises the administration of a purified antibacterial oligonucleotide having about 8 to about 80 nucleotides to the animal in an amount sufficient to inhibit bacterial growth, alleviate a symptom of the infection, or in an amount effective for treatment .
  • the purified antibacterial oligonucleotides of the present invention will preferably bear an enhanced ability to inhibit the growth of bacterial cells relative to previously disclosed oligonucleotide preparations
  • the present invention also represents the first disclosure of the use of oligonucleotides to inhibit the growth of intact clinically relevant bacteria.
  • the oligonucleotides generally inhibit bacterial growth by acting as antisense or antigene inhibitors of bacterial gene expression (when targeted to bacterial nucleic acid sequences) , or by acting aptamerically to alter the function of specific bacterial proteins or polypeptides (when associating target ammo acid sequences contained in bacterial peptides, polypeptides, and proteins) .
  • the oligonucleotides are targeted to an antibiotic resistance gene to render the bacteria sensitive to a conventional antibiotic.
  • the antibacterial oligonucleotides are substantially nuclease resistant (i.e., resistant to nuclease activity) .
  • antibacterial oligonucleotides that have been produced by a process that enhances the oligonucleotide' s antibacterial activity.
  • the presently described antibacterial oligonucleotides will be produced, or otherwise purified, by a process comprising either individually or in combination ion exchange or reverse phase chromatography, extractions, precipitations, gel filtrations, dialysis, diafiltration or functional equivalents.
  • Column chromatography may be by traditional of methods or High- Performance Liquid Chromatography (HPLC) , fast performance liquid chromatography (FPLC) , and the like.
  • the oligonucleotides may be purified by processes including, for example, extraction or precipitation with alcohols or organic solvents.
  • the present invention further contemplates the use of the described antibacterial oligonucleotides, in conjunction with an acceptable pharmaceutical carrier, to prepare medicinal compositions for the treatment of bacterial infections in animals, and more preferably mammals, including humans .
  • Figure 1 shows a dose response curve of different concentrations of antibacterial oligonucleotide NBT 89 (SEQ ID NO. 61) when tested against Escheri chia coli ATCC accession No. 25922.
  • Figure 2 provides a nonexhaustive graph of the types of bacterial genes which proved susceptible to inhibition by antibacterial oligonucleotides.
  • the ordinate shows the categories of bacterial genes defined in Table 2 (A-W) .
  • Figures 3(a-c) show the percent inhibition of the growth of the indicated target bacteria after addition of the indicated oligonucleotide as a function of time.
  • Figures 4 (a-c) show the percent inhibition of the growth of the indicated target bacteria after addition of the indicated oligonucleotide as a function of time.
  • Figures 5 show the percent inhibition of the growth of the indicated target bacteria after addition of the indicated oligonucleotide as a function of time.
  • Figures 6(a-t) are plots of log bacterial growth (and accompanying control cultures) as a function of time after the addition of the indicated oligonucleotide (i.e., "NBT 114" indicates oligonucleotide sequence 114 (SEQ ID NO. 112) from Table 1, infra) .
  • a clinical isolate of Escherichia coli ATCC accession No. 35218 (multiple drug resistant) was used in the experiments corresponding to figures 6 (a-t) .
  • Figures 7(a-j) are plots of log bacterial growth (and accompanying control cultures) of the penicillin resistant clinical isolate of Staphylococcus aureus ATCC accession No. 13301 as a function of time after the addition of the indicated oligonucleotide.
  • Figures 8 shows that animals challenged with the bacterial pathogen Escherichia coli show a significant increase in survival after treatment with oligonucleotide 114 (SEQ ID NO. 112) relative to nontreated control animals.
  • Figure 9 shows that test animals infected with the bacterial pathogen Staphylococcus aureus show a significant increase in survival after treatment with the variant of oligonucleotide 114 (SEQ ID NO. 112), SOT 114.21, relative to nontreated control animals .
  • Figures 10 (a-b) show the results observed when the indicated antibacterial oligonucleotides were tested for bactericidal activity against Staphyl coccus aureus using a standard overnight MIC assay.
  • Figures 11 (a-b) show the results observed when the indicated antibacterial oligonucleotides were tested for bactericidal activity against Serra tia l iquefaciens using a standard overnight MIC assay.
  • Figure 12 shows the results obtained when the indicated antibacterial oligonucleotides were tested using a standard MIC assay against Staph . aureus .
  • Figure 13 shows the results obtained when a variety of different length versions of the indicated antibacterial oligonucleotide were tested using a standard MIC assay against Staph . aureus .
  • Figure 14 shows the results obtained when drug sensitive and drug resistant Staph . aureus were treated with oligonucleotide 114, and ampicillin.
  • Figure 15 shows the results of a standard MIC assay using oligonucleotide MMT 114.15 against P . aeroginosa strain 10145.
  • Figure 16 shows the results of a standard MIC assay using SOT 114.21 against Strep , pyogenes strain 14289. 4.0. DETAILED- DESCRIPTION OF THE INVENTION
  • the present invention describes a method for generating oligonucleotides having the novel property of being capable of having bacteriostatic or bactericidal effects on clinically relevant bacterial pathogens.
  • the oligonucleotides generated using the presently described methods are contemplated to be able to exert antibacterial effect both in vi tro and in vivo .
  • the antibacterial oligonucleotides will be targeted to bacterial sequences where, after associating with or binding to the target sequence, the oligonucleotide disrupts the normal function of the target sequence.
  • the antibacterial effect of the oligonucleotide may be caused by either specific or nonspecific association as long as bacterial growth is inhibited.
  • particularly preferred embodiments of the present invention include the novel antibacterial oligonucleotides, methods of making the antibacterial oligonucleotides, and methods of using the novel antibacterial oligonucleotides to treat bacterial infection.
  • an additional embodiment of the presently described invention is the use of the presently described antibacterial oligonucleotides to treat immunocompromised patients.
  • the antibacterial oligonucleotides may be used to treat bacterial infections m conjunction with similarly engineered antiviral oligonucleotides that are directed to any of a wide variety of human viruses including, but not limited to, adenovirus, human immunodeficiency virus, human leukemia virus, rhino virus, herpes virus, human papilloma virus, respiratory syncytial virus, cytomegalo virus, Epstein bar virus, hepatitis virus (A, B, C and delta), etc.
  • adenovirus human immunodeficiency virus
  • human leukemia virus human leukemia virus
  • rhino virus rhino virus
  • herpes virus human papilloma virus
  • respiratory syncytial virus cytomegalo virus
  • Epstein bar virus Epstein bar virus
  • hepatitis virus A, B, C and delta
  • an additional embodiment of the presently described invention are mixed oligonucleotide compositions that comprise both antiviral and antimicrobial (e.g., antifungal, antibacterial, antiparasitic, etc.) oligonucleotides
  • antiviral and antimicrobial e.g., antifungal, antibacterial, antiparasitic, etc.
  • the relative ratios of the oligonucleotides present in such compositions shall be adjusted to target bacterial, parasitic, fungal, yeast, and viral pathogens that are generally associated as secondary infectious sequelae of infection by one another
  • An additional embodiment of the present invention are therapeutic oligonucleotides that fuse one or more sequences with known antimicrobial, antibacterial, or antiviral therapeutic activity. Such fusions are deemed to constitute novel compositions having broad spectrum activity against multiple and distinct bacterial species, as well as broad antiviral and antibacterial activities Similarly, oligonucleotides bearing multiple active sequences, or mixed compositions of antibacterial oligonucleotides, may be used to target the activity of a gene product in an pathogen by blanket targeting of the DNA (via triplex inhibition, disrupting DNA replication, etc.) and RNA (via RNase H activation or directly disrupting translation, etc.) encoding the activity of interest, as well as by aptameric inhibition of the gene product .
  • the antibacterial oligonucleotides are preferably administered in a pharmaceutically acceptable carrier, via oral, mtranasal, rectal, topical, mtrape ⁇ toneal , intravenous, intramuscular, subcutaneous, lntracramal , subdermal , transdermal, intrathecal methods, or -the like.
  • the preferred formulation for a given antibacterial oligonucleotide is dependent on the location of the target organism in the host animal or the location in a host where a given infectious organism would be expected to initially invade.
  • topical infections are preferably treated or prevented by formulations designed for topical application
  • systemic infections are preferably treated or prevented by administration of compositions formulated for parenteral administration
  • pulmonary infections may be treated both parenterally and by direct application of the antibacterial oligonucleotides to the lung by inhalation therapy.
  • oligonucleotides can often accumulate at relatively high levels in the kidneys, liver, spleen, lymph glands, adrenal gland, aorta, pancreas, bone marrow, heart, and salivary glands. Oligonucleotides also tend to accumulate to a lesser extent in skeletal muscle, bladder, stomach, esophagus, duodenum, fat, and trachea. Lower still concentrations are typically found in the cerebral cortex, brain stem, cerebellum, spinal cord, cartilage, skin, thyroid, and prostate (see generally Crooke, 1993, Antisense Research and Appli cations, CRC Press, Boca Raton, FL) .
  • the presently described antibacterial oligonucleotides can be used to target bacterial infections in specific target organs and tissues.
  • an undesirable symptom e.g., symptoms related to the presence of bacteria in the body
  • the terms "treatment”, “therapeutic use”, or “medicinal use” used herein shall refer to any and all uses of the claimed antibacterial oligonucleotides which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • animal hosts that may be treated using the oligonucleotides of the present invention include, but are not limited to, invertebrates, vertebrates, birds (such as chickens and turkeys, etc.) fish, mammals such as pigs, goats, sheep, cows, dogs, cats, and particularly humans.
  • an appropriate dosage of an antibacterial oligonucleotide, or mixture thereof may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies in normal subjects help establish safe doses. Additionally, therapeutic dosages may also be altered depending upon factors such as the severity of infection, and the size or species of the host .
  • antibacterial oligonucleotides may also be complexed with molecules that enhance their ability to enter the target cells.
  • molecules include, but are not limited to, carbohydrates, polyamines, a ino acids, peptides, lipids, and molecules vital to bacterial growth.
  • the antibacterial oligonucleotide may be complexed with a variety of well established compounds or structures that, for instance, further enhance the in vivo stability of the oligonucleotide, or otherwise enhance its pharmacological properties ( e . g . , increase in vivo half-life, reduce toxicity, etc.) .
  • oligonucleotides are advantageous as an approach to treating bacterial infection because sequences can be specifically designed to inhibit bacterial growth while not interfering with the metabolism of mammalian cells .
  • the present invention also relates to oligonucleotides that have demonstrated antibacterial activity in vi tro .
  • the oligonucleotides will have antibacterial activity as measured in a MIC (minimal inhibitory concentration) test that is recognized in the art as predictive of in vi vo efficacy for the treatment of a bacterial infection with antibiotics.
  • the oligonucleotides of the present invention are able to hybridize to a targeted region of a chosen bacterial polynucleotide (DNA or RNA) to effectively inhibit the ability of that polynucleotide to serve as a template for synthesis of its encoded product (DNA, RNA or protein) , or otherwise inhibit the target sequence's normal function m the bacterium, thereby causing a bacteriostatic or bactericidal effect .
  • a chosen bacterial polynucleotide DNA or RNA
  • Certain oligonucleotides may exert their bacteriostatic or bactericidal effects through binding to and inhibition of protein (aptameric effects) .
  • the invention uses oligonucleotides that are substantially nuclease resistant .
  • Additional methods of rendering an oligonucleotide nuclease resistant include, but are not limited to, covalently modifying the pu ⁇ ne or pyrimidine bases that comprise the oligonucleotide.
  • bases may be methylated, hydroxymethylated, or otherwise substituted (glycosylated) such that the oligonucleotides comprising the modified bases are rendered substantially nuclease resistant .
  • the present invention further relates to compositions comprising nuclease resistant antibacterial oligonucleotides. These compositions generally comprise the oligonucleotide (or a mixture of oligonucleotides) and a physiologically acceptable carrier.
  • the oligonucleotides After administration, the oligonucleotides enter the bacterial cell and bind to the target.
  • the target may be a polynucleotide where hybridization to the oligonucleotide results m an inability of the polynucleotides to serve as templates for their encoded products.
  • the target is a protein
  • the bound oligonucleotide protein complex is inhibited relative to normal protein function (aptameric effect) .
  • growth of the bacteria are inhibited and the effects of the bacteria on the animal are less than they would have been if the oligonucleotides had not been administered
  • the presently described antibacterial oligonucleotides may be formulated with a variety of physiological carrier molecules.
  • the antibacterial oligonucleotides may be combined with a lipid (or cationic lipid) , the resulting oligonucleotide/lipid emulsion, or liposomal suspension may, inter alia , effectively increase the in vi vo half-life of the oligonucleotide.
  • cationic, anionic, and/or neutral lipid compositions or liposomes is generally described in International Publications Nos. WO 90/14074, WO 91/16024, WO 91/17424, Pat. No. 4,897,355, herein incorporated by reference .
  • the antibacterial oligonucleotides of the present invention may also be introduced into bacteria after being complexed with cationic lipids such as DOTMA (which may or may not form liposomes) which complex is then contacted with the target cells.
  • cationic lipids include, but are not limited to, N- (2 , 3 -di ( 9- (Z) -octadecenyloxyl) ) -prop- 1-yl- N,N,N-tr ⁇ methylammon ⁇ um (DOTMA) and its salts, l-O-oleyl-2-O- oleyl-3 -dimethylaminopropyl - ⁇ -hydroxyethylammonium and its salts and 2,2-b ⁇ s (oleyloxy) -3 - (tnmethylammonio) propane and its salts.
  • the antibacterial oligonucleotides may be targeted to specific bacterial cell types by the incorporation of suitable targeting agents ⁇ i . e . , specific antibodies or receptors) into the oligonucleotide/lipid complex.
  • the presently described purified oligonucleotides may be complexed with additional antibacterial agents
  • the described nuclease resistant antibacterial oligonucleotides may also be linked to a conventional antibiotic or other chemical group that inhibits bacterial gene expression
  • antibacterial oligonucleotides are also contemplated to be effective in comoat g bacterial contamination of laboratory cultures, consumables (food or beverage preparations), or industrial processes.
  • oligonucleotide typically refers to a molecule comprising from about 8 to about 80 nucleotides, preferably about 15 to about 35 nucleotides, including polymers of ribonucleotides, deoxyribonucleotides, or both, with the ribonucleotide an ⁇ /or deoxyribonucleotides being connected together via 5 ' to 3 ' linkages that may include any of the linkages known the oligonucleotide art (including, for example, oligonucleotides comprising 5' to 2' linkages)
  • longer oligonucleotides display enhanced targeting specificity but may be less efficient gaming entry to the target bacterium
  • shorter oligonucleotides may more easily permeate the target bacteria, but may display a tendency to nonspecifically associate with host sequences and create a bystander effect or have no effect at all.
  • shorter oligonucleotides may less efficiently bind to, and thus nonspecifically inhibit, bacterial target sequences
  • shorter antisense oligonucleotides (6mers to 7mers) may prove less efficient at specifically binding the target mRNA, and may prove less efficient at activating RNase H activity.
  • Shorter oligonucleotides may also effect host gene expression in a nonspecific, and thus undesirable, manner.
  • the present application additionally contemplates relatively short oligonucleotide sequences (6mers to 7mers) having the desired antibacterial effects, and preferably broad-spectrum antibacterial effects, while exhibiting few adverse side effects in the host.
  • an example of a short (6mer) oligonucleotide is provided below that exhibits significant antibacterial activity and is contemplated as a specific example of a preparation of an antibacterial oligonucleotide that functionally defines the lower size limit of the present invention.
  • the short oligonucleotides of the present invention specifically exclude short inoperative oligonucleotides such as AGGAGGT or GGAG .
  • additional embodiments of the present invention include relatively short (e.g.
  • oligonucleotides that have been identified by using the presently disclosed methods of synthesis in conjunction with standard antibacterial assays while gradually deleting bases from oligonucleotides with established antibacterial activity in order to define short antibacterial "core" sequences.
  • a particular embodiment of the present application contemplates oligonucleotides that have been modified to enhance the specificity of binding. Increased specificity allows for shorter oligonucleotides having the desirable features of both long and short oligonucleotides.
  • oligonucleotides may be constructed using either conventional bases (adenosine, cytosine, guanosine, thy ⁇ nidine, xanthine, inosine, or uridine) or any other modified bases, or base analogues that allow an oligonucleotide comprising such analogues to retain its ability to hybridize to a complementary nucleotide sequence.
  • bases adenosine, cytosine, guanosine, thy ⁇ nidine, xanthine, inosine, or uridine
  • base analogues that allow an oligonucleotide comprising such analogues to retain its ability to hybridize to a complementary nucleotide sequence.
  • non-naturally occurring bases that are capable of forming base-pairing relationships with naturally occurring nucleotide bases include, but are not limited to, aza and deaza pyrimidme analogues, aza and deaza purme analogues as well as other heterocyclic base analogues, wherein one or more of the carbon and nitrogen atoms of the pur e and pyrimidme rings have been substituted by heteroatoms, e . g . , oxygen, sulfur, selenium, phosphorus, and the like.
  • Modified oligonucleotides, nuclease resistant oligonucleotides, and antisense oligonucleotides are also meant to be encompassed by this definition.
  • modified oligonucleotide refers to oligonucleotides that include one or more modifications of the nucleic acid bases, sugar moieties, mternucleoside phosphate linkages, as well as molecules having added substituents , such as diammes, cholesteryl or other lipophilic groups, or a combination of modifications at these sites.
  • the mternucleoside phosphate linkages can be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester , acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phospnonate, phosphorothioate, methylphosphonate , phosphorodithioate , bridged phosphorothioate and/or sulfone mternucleotide linkages, or 3' -3' or 5' -5' linkages, and combinations of such similar linkages (to produce mixed backbone modified oligonucleotides)
  • the modifications can be internal or at the end(s) of the oligonucleotide molecule and can include additions to the molecule of the mternucleoside phosphate linkages, such as cholesteryl
  • Ele ⁇ trophilic groups such as ribose- dialdehyde could covalently link with an epsilon amino group of the lysyl-residue of such a protein.
  • a nucleophilic group such as n-ethylmaleimide tethered to an oligo er could covalently attach to the 5' end of an mRNA or to another electrophilic site.
  • modified oligonucleotides also includes oligonucleotides comprising modifications to the sugar moieties such as 2 ' -substituted ribonucleotides, or deoxyribonucleotide monomers, any of which are connected together via 5' to 3 ' linkages.
  • modified oligonucleotide is meant to encompass all of the foregoing, unless the context dictates otherwise, and also refers to oligonucleotides comprising chemical groups (e.g., sugar molecules, amino acids, etc.) that may improve the antibacterial activity of the oligonucleotide.
  • chemical groups e.g., sugar molecules, amino acids, etc.
  • oligonucleotide backbone refers to any and all means of chemically linking nucleotides such that oligonucleotides result that are capable of base-pairing or otherwise hybridizing, or interacting with a bacterial target sequence in a more-or-less sequence specific manner.
  • purified oligonucleotide refers to an oligonucleotide that has been isolated so as to be substantially free of, inter alia , incomplete oligonucleotide products produced during the synthesis of the desired oligonucleotide.
  • a purified oligonucleotide will also be substantially free of contaminants which may hinder or otherwise mask the antibacterial activity of the oligonucleotide.
  • an oligonucleotide is able to bind to, or gain entry and inhibit the growth of a bacteria, it shall be deemed as substantially free of contaminants that hinder antibacterial activity.
  • One example of a method to produce such purified oligonucleotides is described herein.
  • an oligonucleotide preparation shall generally be considered substantially free of adverse contaminants (e.g., contaminants that hinder the measured antibacterial activity of the nucleotides such as alkyl amines, alkyl ammonium groups, or agents that block oligonucleotide entry, etc.) when the sample proves effective m an m vi tro MIC assay to an extent that is displays more than about twice, and preferably about five times, and most preferably at least about an order of magnitude greater antibacterial activity than a corresponding preparation that has not been treated to remove the adverse contaminants
  • an oligonucleotide preparation shall preferably be considered substantially free of adverse contaminants when the levels of contaminants in a sample are reduced to about l/20th of the levels found in unpurified (or intermediately purified) samples, more typically about l/50th of the levels found m unpurified samples, and preferably less than about 1/lOOth of the levels found intermediately or unpurified samples of oligonucleotide.
  • an antibacterial oligonucleotide preparation may generally be considered free of adverse contaminants when the composition is about 95 percent free, and specifically about 99 percent free of contaminating alkyl amines, alkyl ammonium groups, or a mixture thereof as compared to unpurified crude or intermediately purified samples of the oligonucleotide preparation (as measured by conductivity, mass spectroscopy , or the extent to which a given oligonucleotide preparation retains antibacterial activity) .
  • substantially nuclease resistant refers to oligonucleotides that are resistant to nuclease degradation, as compared to unmodified oligonucleotides, and include, but are not limited to oligonucleotides with modified backbones, such as, for example, phosphorothioates, methylphosphonates, ethylphosphotriesters, 2 ' -O-methylphosphorothioates , 2'-0- methyl -p-ethoxy ribonucleotide, 2'-0-methyl ribonucleosides , methyl carbamates, and methyl carbonates, inverted bases or chimeric versions of these backbones Typically, the relative nuclease resistance of an oligonucleotide will be measured by comparing the percent digestion of a resistant oligonucleotide with tne percent digestion of its unmodified counterpart ( .e., a corresponding oligonucleotide with "
  • Such nuclease resistance tests generally add a given concentration of oligonucleotide (e.g., about 121 ⁇ olar) to a given amount of nuclease Si (at about 0.05 units per ml final concentration in the reaction), Pl (at about 0.05 units per ml final concentration in the reaction), SVP (at about 0.05 units per ml final concentration in the reaction) , Micrococcal Nuclease (at about 0.5 units per ml final concentration in the reaction), etc., and measure the percent degradation (all reactions are incubated at about 37°C in the buffer appropriate for each nuclease.
  • oligonucleotide e.g., about 121 ⁇ olar
  • SI nuclease digestion conditions are typically 30 rnM sodium acetate (pH 4.5), 50 mM NaCl, 1 mM ZnCl,, 5% Glycerol ; Pl nuclease digestion conditions are typically 30 mM sodium acetate (pH 5.3), 0.2 mM ZnCl 2 ; SVP digestion conditions were 100 mM Tris (pH 8.9) 100 mM NaCl , 14 mM MgCl 2 ; and Micrococcal nuclease digestion conditions are typically 50 mM sodium borate (pH 8.8), 5 mM NaCl, 2.5 mM CaCl 2 ).
  • Percent degradation may be determined by using analytical HPLC to assess the loss of full length oligonucleotide, or by any other suitable methods (e.g., by visualizing the products on a sequencing gel using staining, autoradiography , fluorescence, etc., or measuring a shift in optical density) .
  • Degradation is generally measured as a function of time.
  • a substantially nuclease resistant oligonucleotide will be at least about 25% more resistant to nuclease degradation than an unmodified oligonucleotide with a corresponding sequence, typically at least about 50% more resistant, preferably about 75% more resistant, and more preferably at least about an order of magnitude more resistant after 15 minutes of nuclease exposure.
  • targeted to a bacterial sequence refers to the fact that the presently described antibacterial oligonucleotides are substantially homologous, otherwise complementary, or capable of associating with a target bacterial sequence.
  • the presently described antibacterial oligonucleotides are able to disrupt or inhibit the normal function of the target sequence, and hence inhibit bacterial cell division.
  • the antibacterial oligonucleotides will associate or bind to the target bacterial sequence and inhibit the function of the se ⁇ uence by an antisense mechanism, an antigene (triplex) mechanism, or by stearic hindrance.
  • oligonucleotides can function through an aptameric mechanism by binding to nucleic acid binding proteins.
  • apta er shall refer to oligonucleotides that are capable of binding or otherwise interacting with peptides, polypeptides, or proteins in a manner that effects the normal function of the peptide, polypeptide, or protein.
  • the oligonucleotides will generally associate with bacterial target sequences with an avidity sufficient to elicit an antibacterial effect, yet weak enough to allow the oligonucleotide to disassociate from the reaction products (e.g., after messenger degradation, etc.) and subsequently target another molecule.
  • One method of reducing the binding avidity, or relaxing the binding specificity, of an oligonucleotide is to truncate, or delete, a portion of the oligonucleotide.
  • another method of relaxing the binding avidity of an oligonucleotide comprises engineering a percentage of miss-match (or more-or-less neutral match, e.g., G-U base pairs) into the antibacterial nucleotide sequence.
  • a percentage of miss-match or more-or-less neutral match, e.g., G-U base pairs
  • an additional embodiment of the presently claimed methods and oligonucleotides are relaxed-specificity antibacterial oligonucleotides which comprise sequence miss-matches (with the corresponding target sequence) of up to about 60 percent, often about 35 percent, and preferably about 20 percent, or less.
  • miss-match shall apply to all Watson and Crick polynucleotide base-pairs, other than A.-T, G:C, and A:U, and the inverses thereof.
  • the maximally tolerated percentage miss-match may vary depending on the G/C content of the oligonucleotide.
  • an A/T-rich sequence may tolerate a fairly high percentage of miss-match where the G/C base pairs have been retained.
  • the amount of sequence miss-match should not be such that undue side effects result in the host .
  • oligonucleotides comprising partially or fully substituted chemical backbones
  • such oligonucleotides may retain the ability to bind target bacterial sequence under physiological conditions although comprising a greater amount of sequence miss-match than may be tolerated by conventional oligonucleotides.
  • An additional embodiment of the present invention is antibacterial oligonucleotides that are capable of inhibiting bacterial growth by cross reacting with a variety of both known and unknown bacterial target sequences.
  • cross reactive antibacterial oligonucleotide shall refer to an oligonucleotide sequence that inhibits bacterial growth by interacting with bacterial sequences that may share less than 100 percent sequence homology, and preferably at least about 50 percent sequence homology, with the oligonucleotide.
  • Examples of such a cross reactive antibacterial activity include: instances where heterologous , similar, and homologous bacterial sequences are bound and affected by an oligonucleotide that is targeted to a related sequence; instances where an antibacterial oligonucleotide is able to interact with bacterial sequences that share a sufficient percentage of otherwise random sequence complementarity (e . g.
  • a "functional equivalent" of the sequences disclosed in the Sequence Listing shall include any oligonucleotides comprising sequence that is at least about 25 percent sequence homologous, preferably about 33 percent sequence homologous, and more preferably at least about 50 percent homologous to any one of SEQ ID NOS 1-176, and demonstrates at least about 30 percent, and preferably at least about 50 percent of the antibacterial
  • Competent cells refers to bacterial cells that have been manipulated in culture or otherwise chemically, osmotically, or thermally modified such that the cells bear an enhanced ability to internalize exogenous nucleic acid.
  • pathogenic bacteria refers to any and all bacteria that are, or have been, associated with clinical symptoms of disease in animals, including humans.
  • wild-type bacteria refers to a bacteria that has not been modified either chemically or genetically in any way whatsoever (other than growth in culture medium) .
  • a wild-type bacteria shall not be genetically modified such that the bacteria has an enhanced permeability to macromolecules or biological polymers or oligomers .
  • antisense oligonucleotide refers to an oligonucleotide that has a sequence that is substantially complementary to a target DNA or mRNA, so that the antisense oligonucleotide will hybridize in a complementary fashion to the DNA or mRNA to form a complex by Watson-Crick base pairing. Generally, the antisense oligonucleotide will bind the complementary target sequence with an avidity, in vivo, sufficient to inhibit the normal function of target sequence.
  • bacteriostatic oligonucleotide refers to oligonucleotides that inhibit or retard the growth of bacteria either in vi tro or in vivo .
  • bacteriaicidal oligonucleotide refers to oligonucleotides that directly, or indirectly, cause the death of bacteria either in vi tro or in vivo .
  • Gram negative bacteria refers to the inability of bacteria to resist decolorization with alcohol after being treated with Gram's crystal violet stain. However, following decolorization, these bacteria can be readily counter-stained with safranin, imparting a pink or red color to the bacterium when viewed by light microscopy. This reaction is usually an indication that the bacterium's outer structure consists of a cytoplasmic membrane (inner), which is surrounded by a relatively thin peptidoglycan layer, which in turn, is surrounded by an outer membrane.
  • Gram negative bacteria include, but are not limited to, Escherichia , Salmonella , Edwardsiella , Arizona, Ci trobacter, Enterobacter, Proteus, Yersinia , Klyvera, Klebsiella , Neiserria , Vibrio, Pasturella , Hae ophilus , Pseudomonas, Moraxella , Eikenella , Fusobacterium, Acidominococcus , Actinobacillus, Cardiobacterium, Serratia, Providencia , Erwinia, Tatumella, Shigella, Branhamella,
  • Aeromonas Francisella , Gardnerella , Alcal genes , Kingella, Agrobacterium, Leptotrichia , Megasphaera , Capnocytophaga , Cromobacterium, Hafnia , Morganella, Pectobacterium, Cadecea, Helicobacter, Morococcus, Pleisiomonas, Bordetella, Brucella , Achromobacter, Flavobacterium, Bacteroides , Veillonella , Strep tobacillus , Pneumococcu ⁇ , and Calymma tobacterium.
  • Gram positive bacteria refers to the ability of bacteria to resist decolorization with alcohol after treatment with Gram's crystal violet stain, imparting a violet color to the bacterium when viewed by light microscopy. This reaction is usually an indication that the bacterium's outer structure consists of a cytoplasmic membrane surrounded by a thick, rigid bacterial cell wall mainly comprised of peptidoglycan (murein) .
  • Gram positive bacteria include, but are not limited to, Aerococcu ⁇ , Listeria , Streptomyces , Actinomadura , Lactobacillus , Eubacterium, Arachnia , Mycobacterium, Peptostreptococcus , Staphylococcus , Corynebacterium, Erysipelothrix, Derma tophi lus, Rhodococcus, Bifodobacterium, Lactobacillus, Streptococcus , Bacillus, Peptococcu ⁇ , Micrococcus , Kurthia , Nocardia , Nocardiopsis, Rothia, Propionibacterium, Actinomyces, Enterococcus, and Clostridia .
  • antibacterial oligonucleotides may be effective against bacteria including, but not limited to, Campylobacter, Spiri ll i um, Borrelia , Treponema , Leptospira , Legionella , and Chlamydia .
  • mycobacterium refers to any and all strains of bacteria drawn from the group comprising: Mycobacterium tuberculosis , Mycobacterium bovis, Mycobacterium l eprae, Mycobacterium avium-intracellulare, Mycobacterium kansasii , Mycobacterium scrofulsceum, Mycobacterium marinum, Mycobacterium fortui tum, Mycobacterium ulcerans,
  • MIC test refers to a National Committee on Clinical Laboratory Standards (“NCCLS”) approved test for determining the minimum inhibitory concentrations ("MIC”) of bacteria by broth dilution. This term includes the use of this test for determining the percent inhibition of bacterial growth by the oligonucleotides of the invention.
  • transport refers to the movement of the oligonucleotides of the invention from outside the bacterial cell across the bacterial cell's outer-structure and into the bacterial cell's cytoplasm.
  • viral infection factor refers to bacterial products which contribute to the pathogenicity of a bacteria, such as, for example, antibiotic resistance factors, toxins (exo- and endo-), adherence factors that recognize host tissues, extracellular receptors, bacterial iron-bmdmg proteins, and surface modifications that allow the bacteria to escape the immune system (e.g., polysaccha ⁇ de coats or capsules) .
  • labeled oligonucleotides refers to oligonucleotides that have been modified to allow a determination of the presence or amount of the oligonucleotide.
  • Typical labels include, for example, radioisotopes , biot , and enzymes (such as luciferase, or ⁇ - galactosidase) .
  • stringent conditions generally refers to hybridization conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium c ⁇ trate/0.1% SDS at 50°C. ; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumm/0.1% Ficoll/0.1% polyvmylpyrrolidone/50 mM sodium phosphate buffer at pH 6 5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt ' s solution, sonicated salmon sperm DNA (50 g/ml) , 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
  • formamide for
  • hybridization conditions are merely provided for purposes of exemplification and not limitation.
  • stringency may generally be reduced by increasing the salt content present during hybridization and washing, reducing the temperature, or a combination thereof.
  • a more thorough treatise of such routine molecular biology techniques may be found in Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Vols . 1-3 (1989), and periodic updates thereof, herein incorporated by reference.
  • the described oligonucleotides may be partially or fully substituted with any of a broad variety of chemical groups or linkages including, but not limited to: phosphoramidates, phosphorothioates; p-ethoxy; alkyl phosphonate; 2 ' -O-methyl ; 2' modified RNA; morpholino groups; phosphate esters; dithioates; 5' thio groups; propyne groups; or chimerics of any combination of the above groups or linkages (or analogues thereof) , or any other chemical modifications that leave the oligonucleotide capable of specifically binding to nucleic acid or protein.
  • chemical groups or linkages including, but not limited to: phosphoramidates, phosphorothioates; p-ethoxy; alkyl phosphonate; 2 ' -O-methyl ; 2' modified RNA; morpholino groups; phosphate esters; dithioates; 5' thio groups; propyne
  • Oligonucleotides, methylphosphonates, and phosphorothioates may be synthesized, using standard reagents and protocols, on an automated synthesizer utilizing methods that are well known in the art, such as, for example, those disclosed in Stec et al . , J. Am. Chem. Soc . 106 :6077-6089 (1984), Stec et al . , J. Org. Chem. 20 (20 ): 3908-3913 (1985), Stec et al . , J. Chromatog. 326 :263-280 (1985), LaPlanche et al . , Nuc. Acid. Res. l ⁇ (22) : 9081-9093 (1986), and Fasman, G.D. Practical Handbook of Biochemistry and Molecular Biology. 1989, CRC Press, Boca Raton, Florida, herein incorporated by reference.
  • nuclease resistant oligonucleotides The principal criteria for designing nuclease resistant oligonucleotides are: (1) retention of sequence-specific base-pairing and triplex-forming interactions (i.e., the ability to associate with bacterial target sequence such that bacterial growth is inhibited); (2) increasing nuclease stability; (3) ease of synthesis and purification.
  • the most common strategies to date have involved neutralizing the charge on the phosphodiester backbone by substitution at, or replacement of, the phosphodiester moiety, conjugating moieties at the 3' and/or 5' terminus, and substitutions at the 2' -position of ribose and deoxyribose.
  • ODN thioformacetal modified oligodeoxynucleotides
  • Oligomers comprised of the modified linkages formed stable duplexes that exhibited a higher Tm (upon binding complementary RNA) than unmodified RNA-RNA duplexes. Oligonucleotides containing the modified linkages are nuclease resistant. It was found that binding of allyl- modified oligomers to A/U rich mRNA sequences (typical of snRNAs) could be improved by incorporating the modified base 2-ammoadenme m the modified probe.
  • oligonucleotide analogues were resistant to exo- and endonuclease degradation.
  • duplexes formed with the analogues were capable of activating RNase H.
  • the authors suggested that the bulky group attached at the Cl' -position stearically masked the phosphodiester linkage from nuclease attack.
  • the synthesis of uniformly modified 2 ' -deoxy-2 ' -fluoro phosphorothioate oligonucleotides is disclosed by Kawasai et al . , J. Med. Chem. 26:831-841 (1993).
  • Additional antibacterial oligomers may be adapted from the polynucleotide binding polymers and backbones described in Pat. NOS. 5,034,506, 5,142,047, 5,166,315, 5,185,444, 5,470,974, and 5,235,033, which are herein incorporated by reference.
  • the modified oligomer binds to complementary sequences containing naturally occurring ⁇ anomers .
  • a 3'-acridine linked ⁇ -anomer was also prepared. This analogue also demonstrated sequence- specific binding.
  • the ⁇ -anomers demonstrated nuclease stability, independently of whether linked to acridine or not.
  • Base-Modified Nucleoside Units The synthesis of a base analogue designed to recognize T-A and G- C Watson-Crick base pairs to facilitate sequence-specific triplex formation is disclosed by Griffin et al . , J. Am. Chem. Soc. 114 : 7976-7982 (1992) .
  • an antibacterial oligonucleotide may profoundly impact its antibacterial activity.
  • the antibacterial activity of an oligonucleotide may be enhanced by at least 60 percent after it has been subject to an appropriate purification protocol . It is particularly important that purification remove contaminants that either obstruct the uptake of the oligonucleotides or mask the antibacterial activity of the oligonucleotides by, for example, stimulating bacterial growth.
  • the antibacterial oligonucleotides of the present invention were purified by chromatography on commercially available reverse phase (for example, see the RAININ Instrument Co., Inc. instruction manual for the DYNAMAX ® -300A, PureDNA'" reverse-phase columns, 1989, or current updates thereof, herein incorporated by reference) or ion exchange media (see generally, Warren and Vella, 1994, "Analysis and Purification of Synthetic Oligonucleotides by High-Performance Liquid Chromatography" , In Methods in Molecular Biology, vol. 26: Protocols for Oligonucleotide Conjugates. S.
  • Oligonucleotides of the invention were dissolved in pyrogen free, sterile, physiological saline (i.e., 0.85% saline) and sterile filtered through 0.2 micron pyrogen free filters .
  • the principal criteria for designing antisense oligonucleotides for treating bacterial infections are: (1) retention of sequence-specific base-pairing and triplex- forming interactions; (2) increasing nuclease stability; (3) increasing the extent or kinetics of entry into the target cell; (4) activating RNase H (while a consideration, a given oligonucleotide ' s ability to activate RNase H is not strictly required to observe antibacterial activity) ; and (5) ease of synthesis and purification.
  • oligonucleotides are capable of specifically inhibiting bacterial growth as long as they remain capable of associating with the target sequence under the relevant conditions.
  • the use of oligonucleotides to degrade RNA simply requires that the oligonucleotide associate (with at least a four base match) with the bacterial RNA long enough to activate RNase H.
  • oligonucleotides that harbor relaxed sequence specificity are deemed sufficient to activate RNase H.
  • applications are contemplated where the antibacterial oligonucleotide provides the desired inhibitory effect although not specifically targeted, or homologous, to a given bacterial gene.
  • Modified oligonucleotides that activate RNAse H are advantageous because such oligonucleotides will hybridize to their target mRNAs and create a substrate that can be digested by RNase H.
  • RNase H digestion destroys the target mRNA, and thus, these oligonucleotides prevent the translation of the target mRNA. Accordingly, protein expression is inhibited either by the enzymatic destruction of the target mRNA, or by the oligonucleotide physically blocking translation (i.e., after the oligonucleotide directly associates with ribosomal sequence) .
  • RNase H activation is a factor in the design of antibacterial oligonucleotides
  • many antibacterial oligonucleotides e.g., ribonucleotides targeting bacterial RNA
  • modified oligonucleotides that are connected by stretches of unmodified phosphodiester linkages comprising at least about four nucleotides to about seven nucleotides should retain the ability to activate RNase H.
  • phosphorothioate ribonucleotides can also activate RNAase H digestion.
  • the differential specificity of mammalian RNase H (minimum of 5 bases) and bacterial RNAase (4 bases) affords a means of selectively targeting bacterial genes that may have strong sequence homology with certain animal genes .
  • modified oligonucleotides that can form triplexes with duplex DNA (antigene oligonucleotides) , and oligonucleotides that can be used as ribozymes .
  • antibacterial oligonucleotides is aptameric oligomers that are capable of effectively mimicking protein domains and exerting an antibacterial effect by directly associating with bacterial proteins or structures. Additionally, antibacterial oligonucleotides may exert a therapeutic effect by specifically binding and deactivating cellular machinery.
  • the presently described oligonucleotides may directly bind ribosomal sequences and 5 inhibit translation by stearically hindering translation initiation, elongation, disassociation, or by directly destabilizing the structure of the bacterial ribosomes .
  • Antibiotic resistance is often caused by the presence of resistance factors that render an antibiotic ineffective.
  • the presently described oligonucleotides may render an otherwise antibiotic resistant organism sensitive to conventional antibiotics. Accordingly, another embodiment of the present invention is the use of antibacterial oligonucleotides in conjunction with
  • Another embodiment of the present invention involves the use of the presently described oligonucleotides to inhibit the expression of genes whose products regulate the replication or transfer of bacterial genes.
  • antibiotic resistance genes or other virulence factors are often encoded by plasmids, antibacterial oligonucleotides targeted against plasmid replication, transfer (by conjugative transfer) , or gene expression are particularly of interest.
  • antibacterial oligonucleotides targeted against plasmid replication transfer (by conjugative transfer) , or gene expression are particularly of interest.
  • oligonucleotides are contemplated that are capable of inhibiting the expression and transfer of genes encoded by transposable genetic elements (e . g. , transposons) .
  • Antisense oligonucleotides which target essential structural genes, metabolic pathway genes, or transport system genes will inhibit the growth of bacterial cells.
  • virulence factors such as, for example,
  • __ genes encoding antibiotic resistance, toxins, adherence and invasion factors, pili or fimbriae, flagella, antigenic variation factors, and iron binding factors, are also preferred targets. These targets should be pathogen specific, and thus oligonucleotides directed against these targets will preferably not harm either host cells, or the normal bacterial flora of the gut. While some bacterial genes are expressed as individual transcripts, many are transcribed as part of a multicistronic unit or operon. Examples include the ribosomal protein operons, such as the str operon and the alpha operon in Escheri chia coli . Where possible operon transcripts are targeted.
  • Disruption of expression of a gene in the operon may also adversely effect the expression of other genes encoded within the same operon (often in operon transcripts the translation of the 5 '-most genes are required for efficient translation of the downstream genes) . In theory this could result in pleiotropic growth effects from a single oligonucleotide sequence.
  • Specific genes and transcripts are targeted on the basis of their function in the cell. For example, the gene for glucose-6 -phosphate dehydrogenase is central to sugar metabolism.
  • a target gene or operon Once a target gene or operon has been selected, a target region within the gene or operon sequence must be selected, for example, the start codon.
  • An analysis of the sequences around the target sequence e.g., 5' untranslated region, start codon, internal sequence feature, termination codon, 3' untranslated region is performed. This analysis generally encompasses a total of about 120 bases that flank the target sequence. This analysis further predicts the secondary structure of the antisense oligonucleotide, and can be performed using commercially available computer software.
  • the extended target sequence is checked for regions of stable secondary structure.
  • the positions of the bases predicted to be involved in the stem-and-loop structures should be marked and the predicted Tm of the structures noted.
  • stem sequences should be avoided where possible.
  • predicted secondary structures with predicted melting temperature of 45°C or less are disregarded in this analysis.
  • a maximum oligonucleotide length is also selected, and the program identifies the clear regions (no stems, or the structures with the lowest melting temperatures) , and also checks the loop melting temperatures for the generated oligonucleotides.
  • Such programs are well known in the art and include, for example, the program OligoTech version 1.0 (Copyright ® 1995, Oligos Etc. Inc. & Oligo Therapeutics Inc. ) .
  • the length of the flanking se ⁇ uence to be analyzed may be increased if an oligonucleotide with a length of greater than 30 bases is selected.
  • the transcription start site and termination site (or any attenuation sequence) are generally the most distal sequences that will be analyzed. On occasion, this may result in an analysis of about 190 or more bases of flanking sequence.
  • Potential oligonucleotide sequences that have high loop melting temperatures may be eliminated by the above analysis. Note that the melting temperatures for the loops obtained for the commercial programs may need to be adjusted for modified oligonucleotides since these oligonucleotides may have altered base pairing avidities.
  • oligonucleotides Several additional characteristics of the oligonucleotides are also considered. Stable secondary structure (potentially stable under physiologic conditions) , runs of a single base (e . g. , 4 or more A's), and sequences that potentially form stable homodimers are also eliminated if possible. (In cases where double-strand oligonucleotide is the desired end result, homodimers may be preferred.) The base composition of the oligonucleotide is also checked. The two or three oligonucleotide sequences that most nearly meet the above criteria are selected. Using these final oligonucleotides, the program analyzes each sequence and notes loop melting temperatures for both the sense and the antisense strands of the candidate sequences. This decreases the possibility of the computer analysis missing a potential problem structure.
  • the candidate sequences are searched for sequence matches in available sequence databases (for example, Genbank) using commercially available search software.
  • the first search is against the bacterial sequence database (s). This allows the identification of other targets that may also be affected by the candidate sequence, and may also indicate which sequences are potentially effective across bacterial genera. Since many different bacterial genera have highly related genetic organizations or related gene sequences, a potential oligonucleotide may be effective against multiple bacterial genera. For example, the sequences of the gyrA genes of Escherichia coli and
  • Salmonella typhimurium are essentially identical near the start codon.
  • bacterial translation occurs simultaneously with transcription, it may be generally preferable to target antisense oligonucleotides to bacterial sequences at or near the Shine-Delgarno site (ribosome binding site) or to the translation start site of the targeted transcript.
  • the second search is versus a database including human/primate sequences. Since these databases are still quite limited (relative to the entire amount of sequence data in the genome) , databases generally including mammalian sequences should be searched. Oligonucleotides that have high specificity matches to relevant mammalian sequences should be eliminated from initial consideration. (Note: that they may be re-included after further evaluation of the possible target sequences.)
  • this method cannot ensure that all potential targets or conflicts are identified.
  • sequence data will allow an experienced practitioner of the art -to identify targets and select oligonucleotide sequences for use in the methods of the invention.
  • the presently described antibacterial oligonucleotides were tested for antibacterial activity in vitro.
  • a given antibacterial oligonucleotide Prior to use in vivo, a given antibacterial oligonucleotide will have demonstrated antibacterial activity in vi tro against a pathogenic bacteria.
  • the in vi tro antibacterial activity of an oligonucleotide will be tested using a standard bacterial inhibition assay, or MIC test (see National Committee on Clinical Laboratory Standards "Performance Standards for Antimicrobial Susceptibility Testing" NCCLS Document M100-S5 Vol. 14, No. 16, December 1994, herein incorporated by reference).
  • Cells that are growing exponentially in vi tro are generally not representative of cells in clinical infections where nutrients may be limited and the cells are dividing slowly or not at all, i.e., the cells are in stationary phase. Starved stationary phase cells undergo a series of morphological and physiological changes that distinguish them from cells in exponential growth. These changes ensure the prolonged survival of the cells by reducing endogenous metabolism and preparing the cells for possibly adverse conditions.
  • the MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in the tubes or microdilution wells as detected by the unaided eye. Viewing devices intended to facilitate reading microdilution tests and recording of results may be used as long as there is no compromise in the ability to discern growth in the wells.
  • the amount of growth in the wells or tubes containing the antibiotic should be compared with the amount of growth in the growth-control wells or tubes (no antibiotic) used in each set of tests when determining the growth and points.
  • the percent inhibition of an oligonucleotide as reported herein was the absorbance at 625 nanometers of a bacterial culture that was treated with the oligonucleotide divided by the absorbance at 625 nanometers (i.e., O.D. 625) of a duplicate cell culture minus oligonucleotide (control); the resulting number was subtracted from 1, and multiplied by 100%. Small variations in the optical density readings at the lower detection limit of the assay may result in calculated inhibitions of greater than 100 percent. It is assumed that these calculations essentially represent 100 percent inhibition.
  • the concentration of target bacteria used in an MIC assay typically far exceeds the systemic concentrations of pathogenic bacteria that, with the possible exception of abscesses, are expected to be found in vivo . While even the presence of a single bacterium in bodily fluids is considered an indication of infection (John J. Sherris, Editor, Medical Microbiology, An Introduction to Infectious Diseases, 2nd Edition, Elsevier, New York 1990) , the precise number of bacteria/ml is not well quantified in human clinical infections (Kjeldfberg and Knight (3rd Edition) , Body Fluids, 5 ASCP Press, 1993) . It is difficult to quantitate bacteria in body fluids as bacteria are constantly cleared by the immune system (Myrvik, Fundamentals of Medical Bacteriology. 1974, Lea & Febiger, Publishers) . In addition, bacteria grow more slowly in vivo than in vi tro, so this slow growth combined
  • the oligonucleotides need only arrest the growth of the bacteria until the immune system is capable of clearance. Furthermore, in an actual clinical situation, the concentration of bacteria/ml would be far lower than 1.5 x 10 6 /ml, which represents a fatal concentration in Wilson's
  • the bacteria were grown over the period of the assays to an O.D. 600 of 0.1 as defined by the NCCLS . This represents approximately 1 x 10 8 concentration of bacteria which represents more bacteria/ml
  • an antibacterial oligonucleotide sequence e . g. , a phosphorothioate ODN
  • an antibacterial oligonucleotide sequence will be tested for antibiotic activity in a mammalian test subject, and preferably a murine test subject.
  • Phosphorothioate ODNs have previously been tested in mammals (mice, rats, rhesus monkeys), and, when properly administered, have not been found to be significantly toxic.
  • ODNs Prior to introduction in vivo, ODNs will be solubilized in sterile saline and serially-diluted to the desired test concentrations in sterile saline. Bacteria .
  • Bacterial pathogens to be used in vivo include, but are not limited to, inter alia , the drug- resistant Escherichia coli ATCC accession No. 25922, and Staph . aureus ATCC accession No. 13301.
  • the target/test bacteria are cultured in vi tro in Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, MD) for 18 hours at 37°C.
  • cultures of a test pathogen will be prepared by suspending colonies grown on solid medium (for example, trypticase soy agar plates) into 70 ml of Mueller-Hinton broth so that a culture with an optical density of about 0.1 at 540 nm results. Appropriate dilutions of the bacterial cells are then prepared in DPBS .
  • CD1 mice or NMRI mice Six- to eight-week-old CD1 mice or NMRI mice, 24-28 g in size, are typically used in these studies.
  • the CD1 strain of mouse has been used in the past for certain studies of infectious diseases and therapeutics ( e . g . , Brogden et al . , (1986); Cavalieri et al . , (1991); Lister and Sanders,
  • Typical animal tests comprise a minimum of about 5-8 animals in each treatment group (1 cage of 5 mice each) in order to demonstrate adequately the statistical reproducibility of a given experimental observation.
  • Typical animal tests comprise a minimum of about 5-8 animals in each treatment group (1 cage of 5 mice each) in order to demonstrate adequately the statistical reproducibility of a given experimental observation.
  • at least about 5 test animals one can compensate for variabilities such as differing growth rates of microorganisms in a given animal and any variables introduced by the repeated handling and injection of the animals.
  • Test animals are typically injected subcutaneously (SC) on the back (intrascapular) with approximately 0.3 ml of bacterial cell suspension in 1.5% liquified sterile tryptose phosphate agar held at 39°C essentially as described- by Hof et ai . (1986) or I. P. with 5% mucin (Lister & Sanders, 1995) .
  • the test animals are treated by administration of a bolus injection of oligonucleotides at, for example, 0, 1.0, 2.5, 5.0 or 10.0 mg/kg (5 separate groups, one dose per group of 12 animals) to determine optimum therapeutic dose of a given antibacterial oligonucleotide.
  • the oligonucleotide is generally administered I. P. in a volume of approximately 0.5 ml of sterile saline, using a sterile 25-gauge needle or through an Alzets pump.
  • the solution comprising the antibacterial oligonucleotide may also be administered I. V., subcutaneously, orally, or by any other means suitable for the given pathogen being tested.
  • bacteremia will be monitored by collecting daily blood samples from two animals from each group. One fully-anesthetized animal from the negative control group (no bacterial infection) will be bled by cardiac puncture and subsequently euthanized. The number of colony forming units (CFU) in the blood samples will then be determined by plating samples on agar and doing bacterial colony assays .
  • the minimum lethal dose for a given bacterial pathogen e . g. , Escherichia coli ATCC accession No. 25922
  • the minimum lethal inoculum is the minimum dose that results in the death of all of the test subjects during the five to seven days post- infection .
  • female NMRJ mice may be used with, for example, Escherichia coli ATCC accession No. 25922, which is known to cause animal death within five to seven days after intra-clavicular injection.
  • the dose of antibacterial oligonucleotide that protects 50% .of the test animals from death is determined as follows. Beginning at various times after injection of the bacterium into the test animals, and continuing for four days thereafter, the antibacterial oligonucleotide (or its control) is injected S.C. into the test animals in about 0.15 ml DPBS at final concentrations that will vary as appropriate for the given assay. For example, about 0.0, 1.0, 2.0, 2.5, and 5.0 mg/kg of antibacterial oligonucleotide may typically be used.
  • test animals Animals surviving for more than five to seven days after initial bacterial inoculation will be maintained an additional seven days, and then euthanized by C0 2 asphyxiation for further study.
  • the test animals are maintained for more extended periods after initial infection in order to assess the long-term efficacy of oligonucleotide treatment.
  • a similar bacterial inoculation and oligonucleotide treatment protocol can be used to determine the kinetics of bacteria clearance from the peripheral blood of bacteremic animals after treatment with antibacterial oligonucleotide.
  • groups of twelve animals each are infected as above with Escherichia coli , and a group of six mice is sham injected with only saline (the control group) .
  • mice are then treated with (a) saline or (b) oligonucleotide, while the control group is only treated with saline.
  • saline aline or oligonucleotide
  • control group is only treated with saline.
  • blood samples are taken, and the number of test pathogen cells per ml of blood is determined by standard dilution and culture methods .
  • the above animal models are merely exemplary of the myriad of animal models that may be used to establish the efficacy of the presently described antibacterial oligonucleotides, and many other modalities for testing the claimed invention are available to one of ordinary skill.
  • the LD SQ of a given pathogen may be established (or previously known) , and the efficacy of the antibacterial oligonucleotide determined, testing whether substantially all of the test animals survive bacterial exposure.
  • immunocompromised animals may also be used, i . e . , nude mice, SCID mice, etc., to study the antibacterial effects of the described oligonucleotides in the absence of a correctly functioning immune system.
  • compositions containing the oligonucleotides of the invention in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e . g. , intravenous, oral, topical, aerosol (for topical or inhalation therapy), suppository, parenteral, or spinal injection.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs, and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets) . Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
  • tablets may be sugar-coated or enteric-coated by standard techniques.
  • Oral dosage forms of antibacterial oligonucleotides will be particularly useful for the treatment of bacterial infections of the gastrointestinal tract and ulcers caused by or associated with bacterial infection (e . g. , Helicobacter pylori infection, and the like) .
  • bacterial infection e.g. , Helicobacter pylori infection, and the like
  • the presently described antibacterial .oligonucleotides may be used to treat hyperproliferative disorders including, but not limited to, Crohn's disease and ulcerative colitis by specifically eliminating the causative or contributory microorganisms from the bacterial flora of the gut.
  • preparations may comprise an aqueous solution of a water soluble, or solubilized, and pharmaceutically acceptable form of the antibacterial oligonucleotide in an appropriately buffered saline solution.
  • injectable suspensions may also be prepared using appropriate liquid carriers, suspending agents, pH adjusting agents, isotonicity adjusting agents, preserving agents, and the like may be employed.
  • Actual methods for preparing parenterally administrable compositions and adjustments necessary for administration to subjects will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington' s Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pa (1980), which is incorporated herein by reference.
  • the presently described oligonucleotides should be parenterally administered at concentrations below the maximal tolerable dose (MTD) established for the antibacterial oligonucleotide .
  • MTD maximal tolerable dose
  • the carrier may take a wide variety of forms depending on the preparation, which may be a cream, dressing, gel, lotion, ointment, or liquid.
  • Aerosols are prepared by dissolving or suspending the oligonucleotide in a propellant such as ethyl alcohol or in propellant and solvent phases.
  • a propellant such as ethyl alcohol or in propellant and solvent phases.
  • the pharmaceutical compositions for topical or aerosol form will generally contain from about 0.01% by weight (of the oligonucleotide) to about 40% by weight, preferably about 0.02% to about 10% by weight, and more preferably about 0.05% to about 5% by weight depending on the particular form employed.
  • Suppositories are prepared by mixing the oligonucleotide with a lipid vehicle such as theobroma oil, cacao butter, glycerin, gelatin, or polyoxyethylene glycols.
  • antibacterial oligonucleotides may be administered to the body by virtually any means used to administer conventional antibiotics.
  • a variety of delivery systems are well known in the art for delivering bioactive compounds to bacteria in an animal. These systems include, but are not limited to, intravenous or intramuscular or intrathecal injection, nasal spray, aerosols for inhalation, and oral or suppository administration.
  • the specific delivery system used depends on the location of the bacteria, and it is well within the skill of one in the art to determine the location of the bacteria and to select an appropriate delivery system.
  • Oligonucleotides were synthesized using commercial phosphoramidites on commercially purchased DNA synthesizers at either 1 ⁇ M or 15 ⁇ M scales using standard phosphoramidite chemistry. Oligonucleotides were deprotected following phosphoramidite manufacturers protocols. Oligonucleotides to be used unpurified were either dried down under vacuum or precipitated and then dried.
  • Sodium salts of oligonucleotides were prepared using the commercially available DNA-Mate (Barrsk ⁇ gen, Inc.) reagents or conventional techniques such as the commercially available exchange resin, e . g. , Dowex (Tradename) , or by addition of sodium salts followed by precipitation, diafiltration, or gel filtration, etc.
  • DNA-Mate Barrsk ⁇ gen, Inc.
  • conventional techniques such as the commercially available exchange resin, e . g. , Dowex (Tradename) , or by addition of sodium salts followed by precipitation, diafiltration, or gel filtration, etc.
  • Oligonucleotide preparations that would be subject to further purification were initially chromatographed on commercially available reverse phase or ion exchange media (preferably, SAX, .strong anion exchange media) such as Source Q made by Pharmacia, Toyopearl super Q made by Tosohaas, Protein Pak made by Waters, Macroprep Q made by BioRad, and the like. Peak fractions were combined and the samples desalted and concentrated by ethanol precipitation, diafiltration, or gel filtration followed by lyophilization or solvent evaporation under vacuum in commercially available instrumentation such as Savant's Speed Vac.
  • the oligonucleotides may also be electrophoretically purified using polyacrylamide gels .
  • Gel filtration media which may be used include Sephadex or Superdex made by Pharmacia, Trisacryl made by BioSepra, BioGel (preferably P-series, or more preferably P4 ) made by BioRad, Toyopearl HW SEC made by Tosohaas, Cellufine made by Amicon, and the like.
  • the gel filtration step may be repeated several times in order to better remove low molecular weight species, and particularly alkyl amines and/or alkyl ammonium compounds, from the oligonucleotide preparations .
  • Cation exchange columns comprising media such as
  • Macroprep S made by BioRad (preferably in the NH 4 * form), Dowex resins, or Amberlite resins are also useful to remove contaminants from antibacterial oligonucleotide preparations.
  • the pH of the eluted oligonucleotide will be increased to about 7-8 using ammonium hydroxide consequential to this step.
  • oligonucleotides e.g., alkyl amines and/or alkyl ammonium compounds
  • Exhaustive butanol extractions, chloroform extraction followed by ethanol washes or multiple ethanol extractions may be used to obtain purified oligonucleotides that retain antibacterial activity.
  • Oligonucleotides to be used in bacterial experiments were dissolved in pyrogen free, sterile, physiological saline (i.e., 0.85% saline), sterile Sigma H,0, and filtered through a 0.45 micron Gel an filter (or a sterile 0.2 micron pyrogen free filter prior to animal studies) .
  • Table 1 contains a list of all oligonucleotide sequences used in the examples . Although the majority of oligonucleotides used in the examples were constructed using a phosphorothioate backbone, unless otherwise noted, it should be understood that any of a wide variety of chemical backbones could be also used to generate oligonucleotides comprising the sequences listed in Table 1.
  • the antibacterial oligonucleotides were tested for inhibition (INH) activity against drug resistant Gram negative ( Escherichia coli ATCC accession No. 35218) and Gram positive ( Staphylococcus aureus ATCC accession No. 13301) microorganisms.
  • the percent inhibition data in Table 1 were averaged and normalized to a concentration of 2 mg/ml .
  • Tables 2 (A-W) provide time course experiments that test the inhibitory activity (against Escheri chia coli ATCC accession No. 35218 or Staphylococcus aureus ATCC accession No. 13301) of the indicated oligonucleotides when present at 2 mg/ml in the culture medium as targeted against genes that represent nearly all known gene classes in bacteria.
  • Table 2A shows the inhibitory effect of oligonucleotide 28 (NBT 28, SEQ ID NO. 1);
  • Table 2B tests oligonucleotide 10 (SEQ ID NO. 17);
  • Table 2C tests oligonucleotide 43 (SEQ ID NO. 34)
  • Table 2D shows the inhibitory effect of oligonucleotide 27 (SEQ ID NO. 45);
  • Table 2E tests oligonucleotide 2 (SEQ ID NO. 120) ;
  • Table 2F tests oligonucleotide 89 (SEQ ID NO. 61) ;
  • Table 2G tests oligonucleotide 103 (SEQ ID NO. 64);
  • Table 2H tests oligonucleotide 132 (SEQ ID NO. 65) ,
  • Table 21 shows the inhibitory effect of oligonucleotide 19 (SEQ ID NO. 66);
  • Table 2J tests oligonucleotide 16 (SEQ ID NO. 72) ;
  • Table 2K tests oligonucleotide 96 (SEQ ID NO. 79) ;
  • Table 2L tests oligonucleotide 21 (SEQ ID NO. 85) ;
  • Table 2M shows the inhibitory effect of oligonucleotide 18 (SEQ ID NO. 95) ;
  • Table 2N tests oligonucleotide 105 (SEQ ID NO. 103) ;
  • Table 20 tests oligonucleotide 46 (SEQ ID NO. 105);
  • Table 2P tests oligonucleotide 114 (SEQ ID NO. 112);
  • Table 2Q tests oligonucleotide 32 (SEQ .ID NO.
  • Table 2R tests oligonucleotide 73 (SEQ ID NO. 124) ;
  • Table 2T shows the inhibitory effect of oligonucleotide 78 (SEQ ID NO. 134) ;
  • Table 2U tests oligonucleotide 71 (SEQ ID NO. 151) ;
  • Table 2V tests oligonucleotide 14 SEQ ID NO. 154);
  • oligonucleotides are effective against a variety of genes.
  • genes involved in energy metabolism sucrose metabolism, fatty acid metabolism
  • cell division DNA replication, cell wall biosynthesis
  • protein synthesis tRNA synthesis, mRNA stability, rRNA synthesis, ribosomal protein, translation factors
  • virulence factors cell wall and membrane synthesis (fatty acid and phospholipid synthesis, lipopolysaccharide synthesis, periplasmic-secretory proteins, transport proteins, outer-membrane proteins), amino acid biosynthesis, nucleic acid synthesis, nitrate reductase, vitamin metabolism, and drug resistance.
  • Figure 2 shows that the described antibacterial oligonucleotides proved effective against a wide variety of genes from both Gram negative and Gram positive bacteria. More specifically, oligonucleotides targeted against bacterial genes relating to: energy metabolism (A); DNA replication (B) ; cell division (C) ; regulatory proteins (D) ; cell wall biosynthesis (E) ; sugar metabolism (F) ; virulence, pill, flagella (G) ; fatty acid metabolism (H) ; mRNA synthesis (I) ; tRNA synthesis (J) , rRNA synthesis (K) ; ribosomal protein synthesis (L) ; protein synthesis (M) ; phospholipid synthesis (N) ; periplasmic/secretory protein synthesis (0) ; regulation and synthesis of transport proteins (P) , amino acid biosynthesis and metabolism (Q) ; lipopolysaccharide synthesis (R) ; purme/pyrimidme biosynthesis and metabolism (S) , outer membrane protein
  • antibacterial oligonucleotides were effective against virtually every major cellular function tested (as determined by the MIC assay) .
  • this invention may be extended to oligonucleotide targets within newly described bacterial sequences
  • Antibacterial oligonucleotides may be constructed with a range of backbones including, but not limited to: phosphorothioates; p-ethoxy oligonucleotides (partially or fully substituted); or 2'-0-methyl oligonucleotides (partially or fully substituted) . Oligonucleotides comprising all of the above backbones have proved equally effective m inhibiting bacterial growth. In view of the effectiveness of oligonucleotides comprising the chemical backbones listed above, chimeric oligonucleotides (comprising mixed backbones) are also deemed to be effective antibacterial agents.
  • oligonucleotides based on the NBT 18 sequence were also capable of inhibiting the growth of two clinically relevant pathogens that have proven resistant to most conventional antibiotics - Escherichia coli clinical isolate ATCC accession No. 35218 (Tables 3A and 3B) , and Staphylococcus aureus clinical isolate ATCC accession No. 13301 (Tables 3C and 3D) .
  • the NBT 18 sequence variations that were tested in Tables 3A and 3B include: A - the NBT 18 sequence with a 2'-0-Methoxy substituted backbone; B - a truncated (12mer, SEQ ID NO.
  • NBT 18 sequence with a phosphorothioate backbone version of the NBT 18 sequence with a phosphorothioate backbone; C - a truncated (15mer, SEQ ID NO. 175) region of the NBT 18 sequence with a phosphorothioate backbone; D - a truncated (15mer) region of the NBT 18 sequence with a phosphorothioate backbone and a 5' amino group; and E - the NBT 18 sequence with a phosphorothioate backbone.
  • the NBT 18 sequence variations that were tested in Tables 3C and 3D include: A - the NBT 18 sequence with a 2'-0-Methoxy substituted backbone; B - the NBT 18 sequence with a p-ethoxy substituted backbone; C - a truncated (12mer) region of the NBT 18 sequence with a phosphorothioate backbone; D - a truncated (15mer) region of the NBT 18 sequence with a phosphorothioate backbone; and E - a truncated (l ⁇ mer, SEQ ID NO. 176) region of the NBT 18 sequence with a phosphorothioate backbone.
  • Tables 3 (A-D) indicate that the observed antibacterial effect was largely a feature of the antisense sequence of NBT 18 instead of the backbone of a given oligonucleotide ( i . e . , nonspecific sulphur effects, etc.). These data further indicate that oligonucleotides comprising less than one half of the full-length (27 base) sequence of NBT 18 retain the ability to inhibit the growth of at least two clinically significant pathogens.
  • a representative number of the antisense oligonucleotides were tested against a wide variety of bacterial species including Streptococcus ( Streptococcus utans (ATCC accession No. 25175)), Streptococcus pyogenes (ATCC accession No. 14289) , Streptococcus pneumoniae or Pneumococcu ⁇ - pneumoniae (ATCC accession No. 39937), and Streptococcus faecali ⁇ or Enterococcu ⁇ faecali ⁇ (ATCC accession No. 19433), Staphylococcus aureus (ATCC accession No. 29213) , Staphylococcus aureus (ATCC accession No.
  • Klebsiella pneumoniae (ATCC accession No. 4352) , Serra tia liquefaci en ⁇ (ATCC accession No. 27592) , Neisseria sicca (ATCC accession No. 9913), Mycobacterium ⁇ megmati ⁇ (ATCC accession No. 19420), Yersinia mollareti (ATCC accession No.
  • Haemophil us vaginali ⁇ (ATCC accession No. 14018), Shigella sp. (ATCC accession No. 11126) , Vibrio fischeri (ATCC accession No. 7744), and Helicobacter mustelae (ATCC accession No. 43772) .
  • Representative data generated with phosphorothioate forms of the oligonucleotides are provided in Tables 4 (A-Z) .
  • antibacterial oligonucleotides nos. 18 (SEQ ID NO. 73) , 39 (SEQ ID NO. 30) , 63 (SEQ ID NO. 130) , 78 (SEQ ID NO. 134), and 73 (SEQ ID NO.
  • SEQ ID NO. 132 was tested against Klebsi ella pneumoniae (Tables 4E and 4F) ; antibacterial oligonucleotides 2 (SEQ ID NO. 50), 4 (SEQ ID NO. 173), 127 (SEQ ID NO. 143), 63 (SEQ ID NO. 130) , and 73 (SEQ ID NO. 124) were tested 0 against Yersinia mollaretti (Tables 4G and 4H) ; antibacterial oligonucleotides 16 (SEQ ID NO. 72), 12 (SEQ ID NO. 80), 20 (SEQ ID NO. 84), 3 (SEQ ID NO. 121), and 15 (SEQ ID NO.
  • antibacterial oligonucleotide 78 (SEQ ID NO. 134) was tested against Haemophil u ⁇ (Table 4R) ; antibacterial oligonucleotides 114 (SEQ ID NO. 112), 10 (SEQ ID NO. 17), 21 (SEQ ID NO. 85), 18 (SEQ ID NO. 73), and 78 (SEQ ID NO. 134) were tested against Mycobacterium (Tables 4S and 4T) ;
  • antibacterial oligonucleotide 78 (SEQ ID NO. 134) was tested against Heli cobacter (Table 4U) ,- antibacterial oligonucleotides 89 (SEQ ID NO. 61), 127 (SEQ ID NO. 143), 132 (SEQ ID NO. 15) , pl27 (SEQ ID NO. 143 with a p-Ethoxy backbone), 1 (SEQ ID NO. 119), and 76 (SEQ ID NO. 127) were tested against Heli cobacter (Table 4U) ,- antibacterial oligonucleotides 89 (SEQ ID NO. 61), 127 (SEQ ID NO. 143), 132 (SEQ ID NO. 15) , pl27 (SEQ ID NO. 143 with a p-Ethoxy backbone), 1 (SEQ ID NO. 119), and 76 (SEQ ID NO. 127) were tested against Heli cobacter (Table 4U) ,- antibacterial oligonucleotides 89 (SEQ ID NO.
  • Table 4Z The data in Tables 4A-Z indicate that the antibacterial oligonucleotides targeted to varying classes of genes are capable of strongly inhibiting the growth of a broad spectrum of bacterial species. No significant
  • Figures 3 (a-c) respectively provide time course data providing percent inhibition as a function of
  • Figures 4 (a-c) respectively provide time course data showing percent inhibition as a function of time for oligonucleotides 39 (SEQ ID NO. 30) , 78 (SEQ ID NO. 134) , and 63 (SEQ ID NO. 130) as measured against Pseudomonas aeruginosa
  • Figures 5 (a-b) respectively provide time course data showing percent inhibition as a function of time for oligonucleotides 73 (SEQ ID NO. 124) and 114 (SEQ ID NO. 112) as measured against Kl ebsiella pneumoniae .
  • any oligonucleotides chosen and prepared in the manner described herein will be equally effective against a given bacterial target .
  • a wide variety of other bacterial pathogens may be treated using the described compositions. A relatively comprehensive review of such pathogens is provided, inter alia , in Mandell et al . , 1990, Principles and Practice of Infectious Disease 3rd. ed . , Churchill Livingstone Inc., New York, N.Y. 10036, herein incorporated by reference .
  • Tables 5A-D show, in ter alia , that oligonucleotides 21 (SEQ ID NO. 156), 68 (SEQ ID NO. 148), and 85 (SEQ ID NO. 106), 112 (SEQ ID NO. 62), and 18 (SEQ ID NO. 73) continue to substantially inhibit the growth of Staphylococcus aureus ATCC accession No. 13301, for at least 25 hours.
  • oligonucleotides 21 SEQ ID NO. 156
  • 68 SEQ ID NO. 148
  • 85 SEQ ID NO. 106
  • 112 SEQ ID NO. 62
  • 18 SEQ ID NO. 73
  • Escherichia coli ATCC accession No. 35218 represents a particularly virulent, multiple drug resistant strain of Escherichia coli .
  • oligonucleotide number 89 SEQ ID NO.
  • the 24 -hour MIC studies were performed essentially as described above with the exceptions that: growth of the target bacteria to reach an OD 525 of 0.1 occurs in approximately 8 hours instead of about 12 to 16 hours; bacterial growth is monitored throughout the experiment as well as at the end-points; and an additional test was conducted that used starved cells as the initial inoculum instead of fresh log cultures (which provided similar antibacterial results) .
  • the MIC test was carried out as described in Section 4.5., supra .
  • the test oligonucleotides received various post-synthesis treatments, and the percent inhibition of the cell culture growth was calculated as described supra . See
  • Oligonucleotides that were subject to: a) gel filtration; b) four butanol precipitations; or c) two butanol extractions, followed by ethanol or chloroform extractions all demonstrated greater than 85% inhibition of the growth of the test bacteria used in the MIC assay (see B, C, E, G, H and I) .
  • Oligonucleotides may also be purified by strong anion exchange (SAX) chromatography, reverse-phase chromatography, strong cation exchange (SCX) chromatography, followed by size exclusion chromatography (SEC) .
  • SAX strong anion exchange
  • SCX strong cation exchange
  • SEC size exclusion chromatography
  • SCX step may be supplemented or replaced by an alcohol (e.g., ethanol, etc.) precipitation step.
  • contaminant examples include, but are not limited to, quaternary amines (particularly alkyl amines and/or alkyl ammonium compounds), acetamide, acetic acid, 2 -cyanoethanol , isobutyramide , isobutyric acid, benza ide, benzoic acid, succinimide, succinic acid, t-butylphenoxyacetamide (or acetic acid) , phenoxyacetamide (or acetic acid) .
  • quaternary amines particularly alkyl amines and/or alkyl ammonium compounds
  • acetamide acetic acid
  • 2 -cyanoethanol isobutyramide
  • isobutyric acid benza ide
  • benzoic acid succinimide
  • succinic acid t-butylphenoxyacetamide (or acetic acid)
  • phenoxyacetamide or acetic acid
  • Contaminants that are particularly important to remove from the oligonucleotide preparations include compounds that directly or indirectly inhibit bacterial uptake of the oligonucleotides, or otherwise mask the antibacterial effects of the oligonucleotides.
  • One way that a contaminant may mask the antibacterial efficacy of an oligonucleotide is by stimulating bacterial growth in a manner that effectively compensates for the antibacterial activity of a given oligonucleotide.
  • an impurity in anion exchange (AX) HPLC- purified modified linkage oligonucleotides has been isolated and partially characterized which stimulates bacterial growth both in vi tro and in vivo .
  • This impurity/stimulatory material is a mixture of small, polar, multialkyl amino or
  • the impurity can be removed and isolated from the oligonucleotide preparations by using a series of desalting steps.
  • the oligonucleotide was concentrated by first loading the pooled
  • oligonucleotides purified in this manner must contain at least two phosphorothioate or p-ethoxy linkages, or some other non-polar modification in order to adequately absorb to the stationary phase.
  • the oligonucleotide solution was concentrated or removed entirely by lyophilization prior to further purification by size-exclusion chromatography (SEC) .
  • SEC size-exclusion chromatography
  • the oligonucleotide was re-suspended in a minimum amount of water prior to application to the SEC column. Since essentially all of the salt from the AX purification was removed by the RP step, the oligonucleotide was dissolved in a relatively small volume of water. This small volume helps maximize resolution in the SEC step.
  • a column was prepared using virgin BioGel P-4 medium or fine particle SEC medium, using a modified manufacturer's procedure to swell the medium.
  • the column used was 45-50 cm long and 2.2 cm diameter.
  • the flow rate was approximately 1- 2 mL/minute.
  • This size column can be used to purify 1,000- 3,000 OD's of modified linkage oligonucleotides that are at least 12 bases in length. If the oligonucleotides have more than 30% phosphorothioate linkages, the maximum loading drops to about 2,000 OD's.
  • Columns and sample sizes may be scaled up as long as a flow velocity of about 30-75 cm/hr is maintained, and the column height remains at least about 40 cm.
  • the oligonucleotides were eluted with water while monitoring the conductivity and the absorbance at 254 nm.
  • the purification may be easily be modified by monitoring at 280 nm, and the like. Collection began when the oligonucleotide concentration became appreciable (as measured by O.D.), and stopped at no later than about 8 minutes after collection began. If, after the conductivity initially rose, it fell and then began to rise again, collection was terminated. It was important to stop collection as described because oligonucleotides collected after this point typically included the stimulatory impurities.
  • the collected oligonucleotide solutions were checked for concentration and lyophilized. Typically, the above protocol resulted in the purified oligonucleotides having the desired antimicrobial activities.
  • Spectroscopic analysis pointed to a comparatively small, simple molecule, or mixture of similar components, that were eluted along with the oligo. These compound (s) coeluted with oligonucleotide during the reverse- phase concentration/desalting process.
  • analysis by electrospray mass spectroscopy of small molecular weight material removed from an oligonucleotide preparation that had been purified on a Waters Protein Pak 40Q revealed complex mixture of amino compounds with the common feature of signals at m/z 58 and m/z 72.
  • the SEC step outlined above was capable of sufficiently removing the impurities to allow the detection of a consistent pattern of antibiotic activity inherent in the presently described purified oligonucleotides. Accordingly, the SEC step provides a process that allowed for the consistent and predictable removal of the stimulatory impurities from the oligonucleotide preparations.
  • oligonucleotides that have been purified using different procedures consistently showed antibiotic effects that were comparable to the oligonucleotides purified as outlined immediately above.
  • the concentration of ethanol required to elute the oligonucleotides from the reverse-phase column was high enough to allow some removal of the low-absorbing high conductivity material prior to the elution of the oligonucleotides.
  • the resolution was not sufficiently clean to allow straight -forward characterization. This separation was not observed with predominantly S-oligonucleotides .
  • the ability of the RP-column to provide any separation may also be affected by the base composition of the oligonucleotides as well as the type of linkages employed to construct the oligonucleotides.
  • the use of ethanol provided more control over the elution process than acetonitrile, which has higher elution power than ethanol. Additionally, the use of ethanol during this step has implications for cGMP validation.
  • Another feature of the RP step is that the great reduction of inorganic salt during the reverse-phase protocol allows for the use of conductivity to monitor peak elution during the SEC separation. If the salt were not removed, the conductivity signal of the impurities would be masked by the signal from the salt, and conductivity would only be useful for monitoring gross system changes.
  • the alkyl amines and/or alkyl ammonium compounds present in the described impurity apparently act as a counter ion to the phosphodiesters and/or associated to the polar portions of the triester groups of the antibacterial oligonucleotides.
  • the impurity material can not be isolated from blank runs of solutions, reagents, and stationary phases used during the described synthesis and purification procedures. Presently, the impurity has only been observed in oligonucleotides that have been AX purified.
  • Antibacterial oligonucleotides 96ss (SEQ ID NO. 79) and 73ss (SEQ ID NO. 124) (the ss denotes that oligonucleotide 73 is targeted to the sense strand) are homologous to the sense strand of the targeted sequences. Oligonucleotides 96ss and 73ss are thought to exert antibacterial activity by acting as antigene sequences that block gene expression by forming a triple-stranded complex (i.e., triplex formation), or, possibly, by directly interacting with bacterial proteins. A time course of the antibacterial activity of oligonucleotides 73ss and 96ss is shown in Table 7.
  • antibacterial oligonucleotides are also capable of inhibiting the growth of a variety of bacteria that are known to be resistant to various
  • Tables 8 (A-C and F) test the inhibitory activity of oligonucleotide 73 (NBT 73 - SEQ ID NO. 124) against clinical isolates of Escherichia coli that are known to be resistant to: streptomycin (8A) ; sulfonamide (8B) ; penicillin (8C) ,- as well as multiple drug resistant
  • Escherichia col i (8F) .
  • Oligonucleotide 114 (SEQ ID NO. 112) also inhibited the growth of Salmonella typhimurium ATCC accession No. 23564 (8D) , Klebsiella pneumoniae ATCC accession No. 4352 (8E), and Staphylococcus aureus ATCC accession No. 29213 (8G) .
  • Tables 9 test the inhibitory activity of oligonucleotide 114 (NBT 114 - SEQ ID NO. 112) against clinical isolates of Escheri chia coli that are known to be resistant to: streptomycin (9A); sulfonamide (9B) ; penicillin (9C) ; as well as multiple drug resistant Escherichia coli
  • Oligonucleotide 114 (SEQ ID NO. 112) also inhibited the growth of Salmonella typhimurium ATCC accession No. 23564 (9D) , Klebsiella pneumoniae ATCC accession No. 4352 (9E) , and Staphylococcus aureus ATCC accession No. 29213 (9G). Additional studies revealed that antibacterial
  • oligonucleotides 114 (SEQ ID NO. 112), 5 (SEQ ID NO. 152), 39 (SEQ ID NO. 30), 43 (SEQ ID NO. 34), 3 (SEQ ID NO. 51), 78 (SEQ ID NO. 134), 12 (SEQ ID NO. 153), 14 (SEQ ID NO. 154), 23 (SEQ ID NO. 158), 24 (SEQ ID NO. 159), 22 (SEQ ID NO. 157), 17 (SEQ ID NO. 83), 20 (SEQ ID NO. 84), 15 (SEQ ID NO.
  • antibacterial oligonucleotides 16 (SEQ ID NO. 82), 18 (SEQ ID NO. 73), 1 (SEQ ID NO. 119), 5 (SEQ ID NO. 152), 17 (SEQ ID NO. 83), 21 (SEQ ID NO. 156), 132 (SEQ ID NO. 15), 11 (SEQ ID NO. 18), 89 (SEQ ID NO. 61), and 2 (SEQ ID NO. 50) all inhibited the growth of penicillin resistant clinical isolates of Staphylococcus aureus ATCC accession No. 13301 for over 400 minutes when present in the culture medium at a concentration of about 0.5-2.0 mg/ml (data are respectively provided in Figures 7(a-j)).
  • Oligonucleotide 14 (NBT 14 - SEQ ID NO. 154) was used to test whether the antibacterial oligonucleotides could also be used to enhance a target bacteria's sensitivity to antibiotics to which the bacteria had previously proven resistant.
  • Table 10 shows the results of a growth inhibition time course experiment where oligonucleotide 14 was tested for the ability to inhibit the growth of Escheri chia coli Y1088 (known to be resistant to ampicillin) in the presence and absence of the indicated concentration of ampicillin (50 ⁇ g/ml, and 250 ⁇ g/ml) .
  • Table 10 indicates that oligonucleotide 14 is capable of significantly restoring ampicillin sensitivity of Escherichia coli Y1088.
  • the assay was conducted essentially as described in section 4.6, supra , and involved a total of 5 mg of oligonucleotide injected (I.P.) over a 2 day period (1 mg of oligonucleotide suspended in 0.5 ml of sterile saline was injected at 1 , 5, 10, 24, and 34 hours post infection) .
  • Figure 9 shows that mice treated with the antibacterial oligonucleotide SOT 114.21 (phosphorothioate GGAACGCGC linked to 2 ' -methoxy riboCATTGGTATATC with an inverted 3' terminal deoxythymidine) had substantially enhanced survival after challenge with lethal quantities (approximately 10 8 cfu in mucin and iron dextran injected i.p. into CD1 mice) of Staph . Aureus .
  • SOT 114.21 can increase the survival of Staph .
  • Aureus challenged test animals by about 81 percent, and increase the survival of E. coli infected test animals by about 95 percent (relative to animals treated with a placebo) .
  • mice treated with oligonucleotide 132 (SEQ ID NO. 15) in vivo had markedly reduced amounts of bacteremia 24 hours after initial exposure to Escherichia coli ATCC accession No. 25922 (prepared and injected as described in Hof et al . , 1986) .
  • This assay was conducted essentially as described in section 4.6, and involved the injection of a total of 2 mg of oligonucleotide (1 mg injected at 6 and 10 hours post infection) .
  • antibacterial assays were conducted using the standard MIC assay Given that 44 percent of all nosocomial infections are caused by Staph . aureus, Streptococcus , or Pseudomonas, these bacteria were used as targets for standard MIC assays.
  • the standard MIC assay was conducted by using 10x13 mm tubes to which 40 ⁇ l of Mueller Hinton Broth (purchased from BBL, obtained through VWR, 3745 Bayshore Blvd., Brisbane, CA 94005) was added.
  • the oligonucleotides (including an oligo dT control) were supplied as lyophilized pellets and dissolved m 200 ⁇ l of sterile tissue culture water (Sigma) , and 200 ⁇ l aliquots of water or dissolved oligonucleotide were then added to the "control" or "oligo test” tubes.
  • Bacterial suspensions were prepared by suspending the organisms m 1.0 ml of sterile-filtered salme (Sigma) at a concentration corresponding to an O.D. 625 of 0 1-0.102. Ten ⁇ l of this suspension was then added to 990 ⁇ l of salme and 500 ul of this mixture was added to both the "control" and "oligo test" tubes (a concentration of approximately 1x10 s bacteria per ml) Sterile saline was added (260 ⁇ l) to each of test tube to reach a total volume of 1 ml, the tubes were vortexed, O.D.
  • the antibacterial oligonucleotides used in the following studies were constructed as follows (5' to 3 ' ) : SOT T12, 12 thymidmes (first six bases phosphorothioate deoxynucleotides, followed by six 2' -methoxy ribonucleotides and an inverted 3' terminal deoxythymidme linked by a 3 '-3' phosphodiester linkage) ; SOT-Cl2 , 12 cytidines (first six bases phosphorothioate deoxynucleotides , followed by six 2'- methoxy ribonucleotides and an inverted 3' terminal deoxythymidine); SOT 89.6 (phosphorothioate deoxyCAT linked to 2 '-methoxy riboGTC with an inverted 3' terminal deoxythymidine); SOT 89.9 (phosphorothioate deoxyCATGT linked to 2 '-methoxy riboCATT with an
  • SOM 1.1 sulphur, 2 ' -methoxyriboA, linked to phosphorothioate deoxyGCAA, linked to 2 ' -methoxyriboCTGTGTAGCCCA, linked to sulphur, 2 ' -methoxyriboTAG, linked to a 3 ' terminal deoxythymidine, SOM 72.1, or SOM 5.1 (sulphur, 2'-methoxyT, 5 linked to phosphorothioate deoxyACTT, linked to 2 ' - methoxyriboGACATATCGGTC, linked to sulphur, 2'- methoxyriboACT, linked to a 3 ' terminal deoxycytidine), and mixtures of SOT (5.15, 78.15, 89.15, and 114.15) or SOT (89.18, 114.15 (phosphorothioate deoxyCGCCAT linked to 2'- 0 methoxyriboTGGTATATC linked to an inverted 3' terminal deoxyth
  • Figures 10a and 10b show the results of standard 5 overnight MIC assays using the indicated oligonucleotides to test for antibacterial activity against Staph . aureus .
  • Virtually all of the oligonucleotides tested (SOT-T12, SOT- C12, SOT 89.(6, 9, and 12), SOC 89.12, SST 89.12, SOT 1.15, SOT 5.15 (phosphorothioate deoxyACATAT linked to 2'- methoxyriboCGGTCACTC linked to an inverted 3' terminal deoxythymidine), and SOT 143.15) significantly inhibited the growth of Staph . aureus (with the exception of the oligo dT string) relative to the control samples.
  • Figures 11a and lib show the antibacterial activity of oligonucleotides DSM 89.18, SOT 78.15 (phosphorothioate deoxyCATTGT linked to 2 ' -methoxyriboTTGTACTCC linked to an inverted 3' terminal deoxythymidine), SOM 114.15, SOT 89.18 (phosphorthioate deoxyCATGTCAT linked to a 2'- methoxyriboTCTCCTTAAG, linked to an inverted 3' deoxythymidine), SOT 89.21, NBT 89.15, NBT 89.12-1 (phosphorothioate deoxyCATGTCATTCTC linked to a 3 ' terminal inverted phosphorothioate deoxythymidine), NMPT 89.12-2 (phosphorothioate deoxyCATGTCATTC linked to 2 '-methyl, p- ethoxy TC, linked to an inverted 3' terminal deoxythymidine); MPT 89.12-4 (
  • Figure 12 shows the level of growth inhibition obtained when the oligonucleotides SOC 89.12, SOB 89.12, MMT 89.12, MPT 89.12, SOPT 89.12, POT 89.12, DSM 89.18, SSM 89.18, NBT 89.15, NBPT 89.12, MMPT 89.12, SOT 89.12, and SOM- 89F ⁇ were tested in a standard MIC assay against Staph. aureus. All of the tested oligonucleotides proved effective at inhibiting 5 the growth of Staph . aureus .
  • Figure 13 shows that several different length variants uV ⁇ fhSQ 89o21 C6.v-k ⁇ 2, 15'-(phosphorothioate deoxyCATGTC linked to 2 '-methoxy riboATTCTCCTT linked to an inverted 3' terminal deoxythymidine) , and 18mers) were able to inhibit the growth 10 of Staph . aureus when they were tested in a standard MIC assay against Staph . aureus .
  • Figures 14 (a and b) compare the antibacterial activities of -the conventional antibiotic ampicillin and SOT 114.21 against isolates of Staph . aureus strains 13301 and 29213.
  • Figure 15 shows that oligonucleotide MMT 114.15 (2'- ⁇ methoxyri oCGCCATTGGTATATC 1 linked to an ' inverted 3' terminal deoxythymidine) proved capable of inhibiting the growth of P. aeroginosa strain 10145, an opportunistic Gram negative, pathogen that has proved resistant to many conventional ?20>"antibiotics, in a standard MIC assay.
  • Figure 16 shows that oligonucleotide SOT 114.21 proved capable of inhibiting the growth of the pathogen Strep , pyogenes strain 14289 in a standard MIC assay.
  • Tsble SA The Effects of Oligonucleotide Purification Method on the Percent Inhibition of Eschericnia coli 35218 (See Section 5.5.)

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Abstract

Cette invention se rapporte à un nouveau procédé qui démontre l'utilisation thérapeutique d'oligonucléotides résistants aux nucléases pour traiter les animaux souffrant d'une infection causée par une bactérie pathogène. Ce procédé consiste à intégrer: (1) des procédés de sélection de l'oligonucléotide correct; (2) la synthèse et la purification d'oligonucléotides résistants aux nucléases, et (3) des procédés d'analyse in vitro d'oligonucléotides antimicrobiens potentiels. Les oligonucléotides décrits peuvent comporter des squelettes, des restes de sucres, des bases ou des mélanges modifiés et ont été soumis à une purification, ce qui produit des oligonucléotides capables d'inhiber la croissance d'un large spectre d'espèces bactériennes cliniquement pertinentes.
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WO2000057890A1 (fr) * 1999-03-31 2000-10-05 Oligos Etc. Inc. Administration par voie pulmonaire d'acides nucleiques protones/acidifies
WO2001019979A1 (fr) * 1999-09-14 2001-03-22 Merck & Co., Inc. Gene murc et enzyme de pseudomonas aeruginosa
WO2001049350A1 (fr) 2000-01-07 2001-07-12 Chiesi Farmaceutici S.P.A. Inhalateur a aerosol
WO2001036625A3 (fr) * 1999-11-18 2002-01-10 Genesense Technologies Inc SEQUENCES OLIGONUCLEOTIDIQUES ANTISENS DERIVEES DES GENES groEL ET groES COMME INHIBITEURS DE MICRO-ORGANISMES
WO2001042457A3 (fr) * 1999-11-29 2002-01-31 Avi Biopharma Inc Procede et composition d'antisens antibacterien
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US6548651B1 (en) 1998-11-11 2003-04-15 Pantheco A/S Modified peptide nucleic acid (PNA) molecules
EP1071826A4 (fr) * 1998-04-13 2003-05-21 Isis Pharmaceuticals Inc Identification de cibles genetiques destinees a etre modulees par des oligonucleotides et formation d'oligonucleotides destines a subir une modulation genique
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US7381402B2 (en) 2004-02-27 2008-06-03 Chiesi Farmaceutici S.P.A. Stable pharmaceutical solution formulations for pressurized metered dose inhalers
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