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WO2015075166A1 - Méthodes et compositions pharmaceutiques pour traiter une infection bactérienne - Google Patents

Méthodes et compositions pharmaceutiques pour traiter une infection bactérienne Download PDF

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Publication number
WO2015075166A1
WO2015075166A1 PCT/EP2014/075231 EP2014075231W WO2015075166A1 WO 2015075166 A1 WO2015075166 A1 WO 2015075166A1 EP 2014075231 W EP2014075231 W EP 2014075231W WO 2015075166 A1 WO2015075166 A1 WO 2015075166A1
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Prior art keywords
sprx
spovg
nucleic acid
subject
aureus
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Svetlana CHABELSKAIA
Brice Felden
Alex EYRAUD
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
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Universite de Rennes 1
Institut National de la Sante et de la Recherche Medicale INSERM
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/10—Production naturally occurring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Definitions

  • the present invention relates to methods and compositions for the treatment of a bacterial infection.
  • Staphylococcus aureus is a human and animal pathogen that has spectacular adaptive capacities to various antimicrobial agents. S. aureus rapidly acquires multiple antibiotic resistances, causing a wide spectrum of nosocomial and community- associated infections, and is thus a major worldwide health problem [1]. Since the spread of staphylococcal resistance to ⁇ -lactams (such as Methicillin), glycopeptide antibiotics have been the most efficient weapons against gram-positive infections, including the problematic methicillin-resistant S. aureus (MRSA). Vancomycin and Teicoplanin glycopeptides antibiotics have a similar mode of action on cell wall synthesis [2,3].
  • glycopeptides do not block enzymes involved in cell wall synthesis, but instead sequester the substrates required for peptidoglycan formation. Resistance to glycopeptides appeared through selective pressure as a result of a horizontal gene transfer from Enterococcus faecalis to MRSA clinical isolates [4]. In 1997, the first MRSA with reduced susceptibility to glycopeptides was reported in Japan [1,5]. In 2002, MRSA with Vancomycin-resistance was already isolated from two patients [1]. Therefore, there is an urgent need to increase our knowledge of the molecular events involved in antibiotic resistance of this serious pathogen, with a necessary focus on the precious glycopeptides used for MRSA infections. In this way, it has been suggested that characterization of new therapeutic compounds against bacterial infection and against bacterial antibiotic resistances may be highly desirable.
  • yabJ-spoVG methicillin and glycopeptide resistance is adjusted by the yabJ-spoVG operon, which is in turn controlled by a consensus nucleotide sequence of the alternative sigma factor ⁇ [6,7].
  • the yabJ-spoVG mRNA codes for two proteins: YabJ, which has unknown functions; and stage V sporulation protein G (SpoVG).
  • SpoVG was initially identified in Bacillus subtilis and shown to be involved in spore formation [8]. However, in nonsporulating bacteria, its mode of action and the molecular mechanisms involved are not known even though, it was shown recently that SpoVG is a site-specific DNA-binding protein [9].
  • SpoVG (but not YabJ) was shown to be the major regulator of the yabJ-spoVG operon [10]. This yabJ-spoVG operon has been proposed as the ⁇ -dependent secondary regulator [6]. In addition to the control of methicillin and glycopeptide resistance, deletion of the yabJ- spoVG operon was shown to be involved in the control of extracellular nuclease, lipase, and protease expressions [10]. It is also involved in capsule formation and in the transcriptional control of cap and esxA [6,1 1]. Micro-arrays performed on yabJ-spoVG deletion in the Newman strain showed that yabJ-spoVG antagonizes the effects of sigma B on the expression levels of several proteins [10].
  • sRNAs small regulatory RNAs
  • S. aureus expresses around 250 sRNAs, most of which have unknown biological functions [13-18].
  • sRNAs control gene expression through various mechanisms, generally at the post-transcriptional level by direct pairing with mRNA targets [19]. These interactions positively or negatively regulate translation and/or the stability of the mRNAs.
  • aureus sRNAs have been shown to be involved in pathogenicity, e.g., RNAIII, which regulates the expression of numerous virulence factors [14,20-22]. Moreover, the sRNAs SprD and Ssr42 have been shown to be involved in S. aureus virulence in animal models of infection [23,24]. In a large-scale analysis of S. aureus, the inventors identified the small RNA SprX (alias RsaOR) [18]. SprX is expressed from the genome of a converting phage containing virulence factors [18].
  • the present invention relates to methods and compositions for the treatment of a S. aureus infection.
  • the present invention relates to oligonucleotides and their use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.
  • the inventors investigated the development of multiple antibiotic resistances by Staphylococccus aureus bacteria, a major cause of opportunistic and nosocomial infections.
  • the inventors reported the first case of an S. aureus sRNA involved in resistance to Vancomycin and Teicoplanin glycopeptides, the invaluable treatments for methicillin-resistant staphylococcal infections.
  • the inventors demonstrated that modifying SprX expression levels in vivo influences Vancomycin and Teicoplanin glycopeptide resistance.
  • the inventors also demonstrated that the SprX sRNA shapes bacterial resistance to glycopeptides resistance by regulating the expression of stage V sporulation protein G (SpoVG), a DNA-binding protein involved in glycopeptide resistance.
  • SpoVG stage V sporulation protein G
  • the inventors then explored the regulatory mechanism at a molecular level, and uncover the functional SprX domain involved.
  • the inventors demonstrated that SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mR A expression levels or affecting YabJ translation. In fact, SprX inhibits SpoVG expression through the direct interaction between the SprX C-rich loop L3 and spoVG ribosomal binding site ofyabJ-spoVG mRNA.
  • This complex prevents ribosomal loading onto spoVG, and specifically inhibits translation of the second downstream gene within the yabJ-spoVG operon without altering the stability of the mRNA operon.
  • the inventors also demonstrated that a mutated SprX that is unable to regulate SpoVG expression loses its influence on antibiotic resistance in vivo, demonstrating that the regulation of glycopeptides sensitivity by SprX involves SpoVG. This emphasizes the importance of sRNA control in S. aureus pathogenesis and is the first time that regulatory RNA has been shown to be involved in antibiotic resistance in a major human pathogen.
  • the present invention relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 111 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.
  • oligonucleotide refers to isolated, synthetic or recombinant oligonucleotides recognizing or targeting SpoVG mRNA.
  • oligonucleotide also relates to the small RNA “sRNA” recognizing or targeting SpoVG mRNA.
  • oligonucleotide also refers to antisense oligonucletotide (ASO) recognizing or targeting spoVG ribosomal binding site of yabJ-spoVG mRNA.
  • ASO antisense oligonucletotide
  • oligonuclotide also refers to SprX sRNA.
  • SprX has its general meaning in the art and refers to small pathogenicity island RNA X [18], an S aureus Small regulatory RNAs (sRNA).
  • sRNA small pathogenicity island RNA X [18], an S aureus Small regulatory RNAs (sRNA).
  • sRNA small pathogenicity island RNA X [18]
  • SpoVG has its general meaning in the art and refers to stage V sporulation protein G [8,9,10].
  • Staphylococcus aureus has its general meaning in the art and refers to a Gram-positive bacterium, a human and animal pathogen [18]. Staphylococcus aureus is a human commensal of skin and nares and an opportunistic pathogen responsible for a wide variety of human infections including superficial skin and wound infections to deep abscesses (endocarditis and meningitis), septicemia or toxin-associated syndromes. S. aureus is a leading cause of nosocomial and community acquired diseases and any stains acquire antibiotic resistance.
  • glycopeptide antibiotic has its general meaning in the art and refers to a class of antibiotic drugs composed of glycosylated cyclic or polycyclic nonribosomal peptides.
  • Glycopeptide antibiotics include but are not limited to vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, and decaplanin (Pootoolal et al, 2002; Van Bambeke et al, 2004).
  • the present invention also relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence that has at least about 80%, or at least about 85%o, or at least about 90%>, or at least about 95%>, or at least about 96%>, or at least about 97%o, or at least about 98%>, or at least about 99%> nucleic acid sequence identity with a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 11 1 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.
  • Nucleic acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST N (Karlin and Altschul, Proc. Natl Acad. Sci. USA 87(6):2264-2268 (1990)).
  • the present invention also relates to an isolated, synthetic or recombinant oligonucleotide consisting of a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 111 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.
  • the present invention also relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence as set forth by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the present invention also relates to an isolated, synthetic or recombinant oligonucleotide consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • oligonucleotides may be obtained by conventional methods well known to those skilled in the art.
  • the oligonucleotide of the invention may be of any suitable type.
  • the one skilled in the art can easily provide some modifications that will improve the clinical efficacy of the oligonucleotide (C. Frank Bennett and Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic PlatformAnnu. Rev. Pharmacol. Toxicol. 2010.50:259-293.).
  • chemical modifications include backbone modifications, heterocycle modifications, sugar modifications, and conjugations strategies.
  • the oligonucleotide may be selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, LNA, oligonucleotide, morpholinos, small regulatory RNAs (sRNAs), tricyclo-DNA-antisense oligonucleotides (ASOs), U7- or Ul- mediated ASOs or conjugate products thereof such as peptide-conjugated or nanoparticle- complexed ASOs.
  • the oligonucleotide may be stabilized.
  • a “stabilized” oligonucleotide refers to an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo -nuclease). Stabilization can be a function of length or secondary structure. In particular, oligonucleotide stabilization can be accomplished via phosphate backbone modifications.
  • the oligonucleotide according to the invention is a LNA oligonucleotide.
  • LNA Locked Nucleic Acid
  • LNA oligonucleotide refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA analogue nucleotides.
  • LNA oligonucleotides, LNA nucleotides and LNA analogue nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos.
  • LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA.
  • oligonucleotide also include "Morpho linos" (phosphorodiamidate morpholino oligomers, PMOs), 2'-0-Met oligomers, tricyclo (tc)- DNAs, U7 short nuclear (sn) RNAs, or tricyclo-DNA-oligoantisense molecules (U.S. Provisional Patent Application Serial No. 61/212,384 For: Tricyclo-DNA Antisense Oligonucleotides, Compositions and Methods for the Treatment of Disease, filed April 10, 2009, the complete contents of which is hereby incorporated by reference).
  • oligonucleotides of the present invention are oligonucleotide sequences coupled to small nuclear RNA molecules such as Ul or U7 in combination with a viral transfer method based on, but not limited to, lentivirus or adeno-associated virus (Denti, MA, et al, 2008; Goyenvalle, A, et al, 2004).
  • the oligonucleotide of the invention can be synthesized de novo using any of a number of procedures well known in the art.
  • oligonucleotide can be produced on a large scale in plasmids (see Sambrook, et al, 1989).
  • Oligonucleotide can be prepared from existing nucleic acid sequences using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Oligonucleotide prepared in this manner may be referred to as isolated nucleic acids.
  • the oligonucleotide of the present invention is conjugated to a second molecule.
  • said second molecule is selected from the group consisting of aptamers, antibodies or polypeptides.
  • the oligonucleotide of the present invention may be conjugated to a cell or bacterial penetrating peptide.
  • Cell penetrating peptides are well known in the art and include for example the TAT peptide (Bechara C, Sagan S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013 Jun 19;587(12): 1693-702; Malhi SS, Murthy RS.
  • oligonucleotide of the present invention may be conjugated to cell penetrating peptide such as YARVRRRGPRGYARVRRRGPRRC (SEQ ID NO: 4) described in Wesolowski D, Tae HS, Gandotra N, Llopis P, Shen N, Altman S.
  • the oligonucleotide-second molecule conjugate is able to target the S. aureus infected cells.
  • the oligonucleotide-second molecule conjugate targets SpoVG.
  • the oligonucleotide-second molecule conjugate is able to target the S. aureus strain.
  • the oligonucleotide of the invention may be used in reducing or eliminating glycopeptide antibiotic resistances in a S. aureus population.
  • a further aspect of the invention relates to the oligonucleotide of the invention for use as a medicament.
  • the present invention also relates to the oligonucleotide according to the invention for use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.
  • the term "subject" denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with S. aureus infections.
  • antibiotic resistance has its general meaning in the art and refers to bacteria able to survive after exposure to one or more antibiotics.
  • antibiotic resistance refers to resistance of a bacterium to an antibiotic treatment to which it was originally sensitive.
  • oligonucleotides of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the oligonucleotide of the invention to the cells and S.
  • the vector transports the nucleic acid to cells and S. aureus population with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, naked plasmids, non viral delivery systems (cationic transfection agents, liposomes, etc .), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the oligonucleotide sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: RNA viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus.
  • retrovirus as for example moloney murine leukemia virus and lentiviral derived vectors
  • harvey murine sarcoma virus murine mammary tumor virus
  • rous sarcoma virus adenovirus, adeno-associated virus
  • SV40- type viruses polyoma viruses
  • Epstein-Barr viruses papilloma viruses
  • the oligonucleotide sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • two or even more oligonucleotides can also be used at the same time; this may be particularly interesting when the oligonucleotides are vectorized within an expression cassette (as for example by U7 or Ul cassettes).
  • the present invention also relates to a combination of a least one oligonculeotide according to the invention with at least one glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof.
  • the glycopeptide antibiotic agent is selected from the group consisting of but not limited to teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin.
  • the present invention also relates to a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to the invention.
  • the present invention also relates to a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof, comprising the step of administering to said subject at least one oligonucleotide according to the invention in combination with at least one glycopeptides antibiotic.
  • the present invention also relates to a method of treating S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to the invention in combination with at least one glycopeptides antibiotic.
  • the inventors also demonstrated that SprX negatively regulates the expression of the general transcriptional factor SpoVG. Accordingly, the present invention also relates to the oligonucleotide according to the invention for use in the treatment of S. aureus infection in a subject in need thereof.
  • compositions and kits of the invention are provided.
  • the oligonucleotide of the invention may be used or prepared in a pharmaceutical composition.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the oligonucleotide of the invention and a pharmaceutical acceptable carrier for use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.
  • the oligonucleotide of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions of the present invention for oral, inhalation, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration of the oligonucleotide, alone or in combination with another active principle can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, inhalation administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and nasal or intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being administered by nasal administration or by inhalation.
  • Nasal administration may be under the form of liquid solution, suspension or emulsion. Solutions and suspensions are administered as drops. Solutions can also be administered as a fine mist from a nasal spray bottle or from a nasal inhaler. Inhalation may be accomplished under the form of solutions, suspensions, and powders; these formulations are administered via an aerosol, droplets or a dry powder inhaler. The powders may be administered with insufflators or puffers.
  • compositions of the present invention include a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc.
  • a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc.
  • the compositions will generally be in the form of a liquid, although this need not always be the case.
  • Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, celluose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, mineral oil, etc.
  • the formulations can also include lubricating agents, wetting agents, emulsifying agents, preservatives, buffering agents, etc.
  • lubricating agents e.g. cationic lipids or neutral lipids, or mixtures of these
  • nucleic acids are often delivered in conjunction with lipids (e.g. cationic lipids or neutral lipids, or mixtures of these), frequently in the form of liposomes or other suitable micro- or nano-structured material (e.g. micelles, lipocomplexes, dendrimers, emulsions, cubic phases, etc.).
  • the amount of an oligonucleotide to be administered will be an amount that is sufficient to induce amelioration of unwanted disease symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition, of the subject, etc. and may be determined on a case by case basis. The amount may also vary according to the type of condition being treated, and the other components of a treatment protocol (e.g. administration of other medicaments such as steroids, etc.). If a viral-based delivery of oligonucleotides is chosen, suitable doses will depend on different factors such as the viral strain that is employed, the route of delivery (intramuscular, intravenous, intra-arterial, oral, inhalation or other).
  • oligonucleotides of the invention will likely be administered on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart.
  • compositions of the invention may include any further glycopeptide antibiotic agent which is used in the treatment of S. aureus infection.
  • pharmaceutical compositions of the invention can be co-administered with glycopeptide antibiotic agent selected from the group consisting of teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin.
  • glycopeptide antibiotic agent selected from the group consisting of teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin.
  • the invention also provides kits comprising at least one oligonucleotide of the invention. Kits containing oligonucleotide of the invention find use in therapeutic methods.
  • a further aspect of the invention relates to a method of identifying a S. aureus infected subject having or at risk of having or developing a glycopeptide antibiotic resistance which comprises the steps of:
  • biological sample refers to sample obtained from S. aureus infected subject such as blood and urine.
  • biological sample also refers to S. aureus strain isolated from S. aureus infected subject.
  • a “reference value” can be a “threshold value” or a “cut-off value”. Typically, a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the person skilled in the art may compare the SprX expression levels obtained according to the method of the invention with a defined threshold value.
  • the threshold value is derived from the SprX expression level (or ratio, or score) determined in a biological sample derived from one or more subjects having a glycopeptide antibiotic resistance.
  • the threshold value may also be derived from SprX expression level (or ratio, or score) determined in a biological sample derived from one or more subjects not having glycopeptide antibiotic resistance.
  • retrospective measurement of the SprX expression levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.
  • the reference value may consist in expression level measured in a biological sample associated with a subject having glycopeptide antibiotic resistance or in a biological sample associated with a subject not having glycopeptide antibiotic resistance.
  • a further aspect of the invention relates to a method for determining whether a S. aureus infected subject is a responder or is a non-responder to a glycopeptide antibiotics treatment which comprises the steps of:
  • responder refers to S. aureus infected subject that will respond to glycopeptide antibiotics treatment.
  • non-responder refers to a subject that will not respond to glycopeptide antibiotics treatment.
  • non-responder also refers to a subject infected with S. aureus strain resistante to glycopeptide antibiotics treatment.
  • Methods for measuring the expression level of SprX in a biological sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a sRNA.
  • the prepared nucleic acid can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFFYMETRIX).
  • the analysis of the expression level of SprX involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in U. S. Patent No.
  • the determination comprises hybridizing the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of SprX.
  • Hybridization may be performed by any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. Nucleic acids exhibiting sequence complementarity or homology to the nucleic acid of interest herein find utility as hybridization probes or amplification primers.
  • nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical.
  • appropriate means such as a detectable label
  • a wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e.g. avidin/biotin).
  • the probes and primers are "specific" to SprX they hybridize to, i.e.
  • SprX may be expressed as absolute expression profile or normalized expression profile.
  • expression profiles are normalized by correcting the absolute expression profile of SprX by comparing its expression to the expression of a nucleic acid that is not a relevant, e.g., a housekeeping nucleic acid that is constitutively expressed.
  • This normalization allows the comparison of the expression profile in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • Probe and or primers are typically labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labelled" is intended to encompass direct labelling of the probe and primers by coupling (i.e., physically linking) a detectable substance as well as indirect labeling by reactivity with another reagent that is directly labeled.
  • detectable substances include but are not limited to radioactive agents or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • Indocyanine Cy5
  • the invention also relates to a kit for performing the methods as above described, wherein said kit comprises means for measuring the expression level of SprX that is indicative of subject having or at risk of having or developing a glycopeptide antibiotic resistance.
  • the kit may include primers or probes as above described.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices,
  • a further object of the invention relates to a method for the treatment of S. aureus infection in a subject in need thereof comprising the steps of:
  • a further object of the invention relates to a glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof, wherein the subject was being classified as responder by the method as above described.
  • a further object of the invention relates to the oligonucleotide according to the invention in combination with at least one glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof, wherein the subject was being classified as non responder by the method as above described.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Alignment of the nucleotide sequences of SprX from the N315 strain and the two SprX copies from the HGOOl strain. Identical nucleotides in the sequences are indicated with asterisks. The nucleotide numbering is based on the SprX2 sequence.
  • Bacterial strains Bacterial strains, plasmids construction, and culture conditions.
  • the bacterial strains and plasmids used in this study are listed in Table I.
  • the bacteria were grown at 37°C in Brain-Heart Infusion broth (BHI, Oxoid) or in Tryptic Soy Broth (TSB, Oxoid). When necessary, the media were supplemented with either 10 ⁇ g of chloramphenicol or ⁇ erythromycin for S. aureus, or 50 ⁇ g ampicillin per mL for E. coli.
  • BHI Brain-Heart Infusion broth
  • TLB Tryptic Soy Broth
  • sprX2 was expressed from its own promoter.
  • the sprX2 sequence (containing the 207-base pair (bp), upstream and 258-bp downstream from sprX) was amplified from HGOOl genomic DNA as a 615-bp fragment.
  • the pCN38-sprX_mutL3 was produced using the mutagenized oligonucleotides 'mutfor' and 'mutrev'. All fragments were flanked with Pstl and EcoBl restriction sites.
  • the PCR products were cloned in pCN38 [37]. For the spot assays, overnight cultures of the tested strains were diluted to obtain a OD600 of 8. From these cultures, seven consecutive ten-fold dilutions were prepared.
  • Table I Strains and plasmids used in this study.
  • the resulting plasmids pBT2AsprXl and pBT2AsprX2 were transformed into S. aureus strain RN4220 and then into S. aureus HG001. Mutants were enriched by cultivation at 42°C. Cells from the stationary phase culture were plated on TSA plates and incubated at 37°C. Colonies were imprinted on plates that were supplemented with 10 ⁇ g/mL chloramphenicol. Chloramphenicol-sensitive colonies were tested by PCR for deletion of sprXl and sprX2. The deletions were confirmed by Northern blot assays.
  • the S. aureus HG001 AyabJ-spoVG: :erm mutant was constructed by transducing the AyabJ-spoVG::erm mutation of strain RN4220 [7] into strain HG001.
  • the deletion of the yabJ-spoVG locus was confirmed by PCR, Northern blot, and Western blot analysis.
  • the pSTM33 vector containing SpoVG-His6 [7] was electroporated into BL21 strain (DE3, Novagen). Cells were grown in LB broth to a 600 nm optical density of 0.5, and SpoVG-His6 expression was induced with 0.3 mM isopropyl-D-thiogalactopyranoside (IPTG, Eurobio). After 3 hours, cells were collected by centrifugation and resuspended in 30 mL phosphatebuffered saline (pH 7.4) supplemented with a cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) and 0.1 mg/mL DNase.
  • IPTG isopropyl-D-thiogalactopyranoside
  • the cells were disrupted and the debris separated by centrifugation at 13,000 g for 10 minutes.
  • Purification of the His-tagged protein was performed on nickel Sepharose High Performance columns (HisTrap HP; GE Healthcare) using an AKTA fast-performance liquid chromatography system. The correct molecular weight of the purified protein was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. One milligram of the purified SpoVG-His 6 was used to raise polyclonal mouse antibodies (Eurogentec, Seraing, Belgium).
  • culture pellets corresponding to 2 mL of culture at a OD600 of 1 were suspended in 0.2 mL of lysis buffer (10 mM Tris-HCl pH 7.5, 20 mM NaCl, 1 mM EDTA, 5 mM MgC12, and cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) containing 0.1 mg/mL lysostaphin. Following incubation at 37°C for 10 minutes, Laemmli sample buffer was added [40]. Samples were then boiled for 5 minutes, separated by SDSPAGE, and transferred onto a hybond-P PVDF membrane (Amersham).
  • the proteins were centrifuged for 15 minutes at 4°C (4,000 g) and washed with 1 mL 80% acetone, then pelleted for 5 minutes at 4°C (4,000 g). The pellets were dried at room temperature. Duplicate pellets were dissolved in 200 urea 8M. 2D-DIGE and mass spectrometry identification of proteins of interest were performed by the Cochin Institute (Paris). Spots corresponding to proteins with expression modified between HG001 wt and AsprX were subjected to in-gel trypsin digestion, peptide extraction, and desalting, followed by MALDI-ToF/ToF analysis. Peptides were analyzed using MASCOT and the NCBlnr database to identify the selected protein spots. The average ratio of protein levels was calculated using DeCyder 2D software (GE Healthcare) to determine the change in normalized spot volume between HG001 wt and HG001 AsprX samples. RNA isolation, Northern blots, transcription and RNA labelling.
  • RNAs were prepared as previously described [18]. For SprX, Northern blots were performed with 10 ⁇ g of total RNA [13]. Specific 32 P-labeled probes were hybridized with ExpressHyb solution (Clontech) for 90 minutes, washed, exposed, then scanned with a Typhoon FLA 9500 scanner (GE Healthcare). For the yabJ-spoVG mRNA, Northern blots were performed [6]. The primer pair oSTM29/oSTM30 was used to generate adCTP-labeled spoVG-specific probes by PCR labeling. For in vitro experiments, RNAs were transcribed from PCR fragments generated from genomic DNA.
  • SprX was prepared by incubating 14 pmol of unlabeled RNA in a buffer (10 mM Tris-HCl pH 7.5, 60 mM NaCl, 1 mM EDTA) for 10 minutes at 25°C.
  • I e yabJ-spo mRNA was prepared by incubating 1 pmol of labeled RNA in the aforementioned buffer.
  • MgCl 2 was added to obtain a final concentration of 2.5 mM and this was then incubated for 10 minutes at 25°C.
  • the toeprint assays were performed as previously described [18], but with modifications. Annealing mixtures containing 13 nM of yabJ-spoVG mRNA with either 67 nM of labeled Toeprint spoVG or Toeprint yabJ primer were incubated with a buffer (20 mM Tris-HCl pH 7.5, 60 mM NH4C1) for 2 minutes at 90°C followed by 1 minute at RT. Renaturation was realized in the presence of 10 mM MgC12 at 25 °C for 20 minutes. For the assays in the presence of SprX, various concentrations of SprX were added prior to the purified E. coli 70S ribosomes.
  • ribosomes were reactivated for 15 minutes at 37°C and diluted in the reaction buffer in the presence of 1 mM MgC12. 33 nM 70S were added in each assay, incubated for 5 minutes and the MgC12 was adjusted to 10 mM. After 5 minutes, 0.83 ⁇ of uncharged tRNAfMet was added and this was incubated for 15 minutes.
  • cDNA was synthesized with 4 UI of AMV RT (Biolabs) for 15 minutes. Reactions were ended by the addition of 15 ⁇ of loading buffer II (Ambion). The cDNAs were loaded and separated onto 8% polyacrylamide/8M urea gels. Sequencing ladders were generated with the same 5'- endlabeled primer. Results
  • SprX is expressed from the genome of a converting phage containing virulence factors [18].
  • SprX is expressed from the genome of a converting phage containing virulence factors [18].
  • the inventors analyzed the phenotypes of strains with different sRNA expression levels. For this purpose, the inventors used HG001 agr+ S. aureus strain carrying two copies of sprX. Alignments of SprXl and SprX2 showed that the RNA sequences are identical in each, except for three nucleotides ( Figure 1).
  • the inventors disrupted the two copies of sprX in strain HG001 (AsprX) by homologous recombination, thus abolishing SprX expression.
  • Overexpression of SprX was achieved using a multicopy plasmid expressing SprX from its endogenous promoter (pCN38- sprX).
  • pCN38- sprX a multicopy plasmid expressing SprX from its endogenous promoter
  • the inventors measured their resistance to Teicoplanin and Vancomycin, two antibiotics from the glycopeptide family.
  • SprX deletion and overexpression had no effect on bacterial growth in Mueller-Hinton (MH) medium.
  • SprX reduces the expression of a protein involved in bacterial resistance to numerous antibiotics.
  • SpoVG is translated from a -1200 nt-long bicistronic yabJ-spoVG mRNA that is cleaved by an unknown mechanism into two transcripts of -600 and -500 nts, fragments which correspond respectively to yabJ and spoVG mRNA.
  • the inventors used a labeled DNA probe of 300 bp encompassing SpoVG open reading frame (ORF) to detect both the spoVG and yabJ-spoVG mRNA. No detectable changes were seen in levels of either mRNA in the AsprX strain when compared to the wt parental strain. Also, no SprX-induced changes were observed for the yabJ mRNA using a ja ⁇ J-specific probe. Collectively, these results show that SprX lowers SpoVG expression, and that regulation occurs at the posttranscriptional level. Structural changes of the yabJ-spo VG mRNA induced by SprX.
  • yabJ-spoVGiei which contains the ribosome binding site (RBS) of spoVG and the first 86 nts of the spoVG ORF.
  • the inventors subjected the yabJ-spoVGiei alone or in complex with SprX to statistical nuclease SI (specific for single-stranded RNAs) and to RNase VI cleavages (specific for double-stranded RNAs).
  • SI single-stranded RNAs
  • RNase VI RNase VI cleavages
  • the presence of many SI cleavages in the region A558-A630 of yabJ-spoVGiei mRNA supports the existence of unpaired single-strand domains.
  • the simultaneous presence of Vi and Si cleavages at the same nucleotide position within Hl-Ll and H2'-L2' may be explained by the coexistence of two alternating structures at the SprX 5 '-end. Hairpins H3-L3 and H4-L4 in the 3 ' region of SprX are however well-supported by the probing patterns.
  • a C-rich nucleotide sequence situated within the SprX L3 loop has been proposed to interact through base pairings with the spoVG SD sequence.
  • these mutational data support the proposed model of SprX-yabJ-spoVG mRNA interaction, which involves pairings between the SprX L3 loop and the spoVG RBS of the yabJ-spoVG mRNA.
  • SprX specifically reduces spoVG translation initiation of the yabJ-spoVG operon mRNA.
  • SprX might also prevent ribosome loading onto the yabJ RBS of the yabJ-spoVG mRNA.
  • One ribosome toeprint was detected 16 nts downstream from the predicted yabJ initiation codon.
  • the addition of increasing concentrations of SprX did not alter the ribosome binding.
  • SprX_mutL3 is able to regulate SpoVG protein expression in vivo.
  • SprX influences glycopeptide antibiotic resistance by regulating SpoVG.
  • deletion of the yabJ-spoVG operon reduces the resistance to glycopeptide antibiotics [7]. Complementation by a vector allowing the expression of only the SpoVG protein was sufficient to restore the antibiotic resistance [7].
  • deletion of yabJspoVG would affect resistance to Teicoplanin and Vancomycin in the S. aureus strain HG001.
  • deletion of the yabJ-spoVG operon reduces bacterial resistance to both antibiotics.
  • SprX_mutL3 (already shown to not influence SpoVG expression) could reduce this resistance. As demonstrated in this report, the overexpression of SprX reduced S.
  • RNA expressed from an S. aureus pathogenicity island We provide evidence that SprX shapes S. aureus resistance to Vancomycin and Teicoplanin glycopeptides, two invaluable antibiotics for treatment of methicillin-resistant staphylococcal infections. This is the first time that a regulatory RNA has been shown to be involved in antibiotic resistance in a major human bacterial pathogen.
  • SpoVG (not YabJ) is the major regulator of the yabJ-spoVG operon [7,10] and on its own it can rescue the phenotypes related to the deletion of the yabJ-spoVG operon.
  • the molecular mechanisms underlying SpoVG action in these biological pathways remain unknown.
  • SpoVG is a DNA- binding protein supporting the hypothesis that it could act as a transcriptional factor [9].
  • SprX affects S. aureus resistance to two glycopeptide antibiotics. Further studies on the regulation of the additional phenotypes related to yabJ-spoVG will be necessary to fully understand the importance of SprX in the regulation of other bacterial processes.
  • SprX interacts by antisense pairings with the spoVG ribosomal binding site (including its SD and AUG initiation codon) of the yabJ-spoVG mRNA.
  • spoVG RBS a C-rich sequence situated in an accessible loop within the SprX structure. This functional sequence contains an UCCC motif, a specific conserved signature that has been detected in several previously-studied S. aureus mRNAs [14]. This reinforces the notion that gene expression regulation by these sRNAs in S. aureus occurs through a shared mechanism.
  • the SprX-yabJ-spoVG mRNA interaction inhibits SpoVG translation initiation by preventing the binding of ribosomes to the spoVG RBS.
  • the translation initiation inhibition is a strategy commonly used by bacterial sRNAs to control gene expression. sRNA pairing with mRNA targets can enhance or repress targeted gene translation [19,28,29]. Generally, duplex formation between the mRNA target and sRNAs induces mRNA target degradation through the recruitment of bacterial ribonucleases [30].
  • SprX inhibits SpoVG translational initiation without inducing yabJ-spoVG mRNA degradation. Such strategy was shown to be sufficient for gene silencing in both Gram-negative and -positive bacteria [23,31].
  • the bicistronic yabJ-spoVG mRNA is cleaved by an unknown mechanism into two separate transcripts that correspond to the yabJ and the spoVG mRNAs.
  • SpoVG cannot be translated from the spoVG mRNA because the mRNA cleavage occurs downstream from its SD nucleotide sequence [7].
  • the physiological functions of the yabJ-spoVG mRNA processing remain to be identified, but this cleavage could serve to irreversibly inhibit spoVG expression.
  • the full-length yabJ-spoVG mRNA and spoVG mRNA are observed at similar levels in both wt and AsprX strains.
  • the inventors measured the levels of the yabJ transcript and determined that SprX has no effect on its amount. This suggests that the internal processing ofyabJ-spoVG mRNA is SprX-independent.
  • sRNA-mediated operon control adds an additional layer to gene expression regulation.
  • Diverse sRNA mechanisms for the adjustment of operon expression have been discovered.
  • sRNA could also influence operon mRNA stability. Indeed, sRNAs could trigger degradation of the entire operon mRNA [32], or of just a part of the operon, releasing a translationally-active mRNA fragment [33].
  • sRNA could also induce operon-mRNA cleavages [34].
  • RNAIII the effector of the global agr regulon that controls the synthesis of multiple virulence factors [14,20-22].
  • RNAIII is produced at the end of the exponential phase and allows for the transition between synthesis of surface-associated proteins and secreted factors [35].
  • Others regulatory sRNAs such as SprD and Ssr42 have been shown to be essential for the virulence of S. aureus in an animal model of infection [23,24].
  • SprD was shown to regulate the expression of an immune evasion molecule (Sbi) secreted by bacteria to impair the host immune responses.
  • an immune evasion molecule (Sbi) secreted by bacteria to impair the host immune responses.
  • a riboswitch (a 5 ' leader sequence within mR A) was shown to control the translation of the mRNA-encoding aminoglycoside adenyl-transferase enzymes that confer resistance to aminoglycoside antibiotics.
  • drug-binding to the mRNA leader releases the translation repression imposed by the riboswitch and induces bacterial resistance to aminoglycosides.
  • RNA 16 2051- 2057.

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Abstract

La présente invention concerne des méthodes et des compositions pour traiter une infection bactérienne. La présente invention concerne des oligonucléotides et leur utilisation dans une méthode pour améliorer l'efficacité clinique d'antibiotiques glycopeptides pour le traitement d'une infection à S.aureus chez un sujet ayant besoin d'un tel traitement.
PCT/EP2014/075231 2013-11-22 2014-11-21 Méthodes et compositions pharmaceutiques pour traiter une infection bactérienne Ceased WO2015075166A1 (fr)

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