WO2014085866A1 - Utilisation d'antagonistes de miarn pour la modulation du système immunitaire, traitement d'infections bactériennes et traitement de maladies des voies aériennes - Google Patents

Utilisation d'antagonistes de miarn pour la modulation du système immunitaire, traitement d'infections bactériennes et traitement de maladies des voies aériennes Download PDF

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WO2014085866A1
WO2014085866A1 PCT/AU2013/001421 AU2013001421W WO2014085866A1 WO 2014085866 A1 WO2014085866 A1 WO 2014085866A1 AU 2013001421 W AU2013001421 W AU 2013001421W WO 2014085866 A1 WO2014085866 A1 WO 2014085866A1
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mir
mirna
subject
expression
sequence
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Paul Stephen Foster
Gerard KAIKO
Hock TAY
Philip Hansbro
Joerg Mattes
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Newcastle Innovation Limited
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Publication of WO2014085866A1 publication Critical patent/WO2014085866A1/fr

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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
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/17—Immunomodulatory nucleic acids
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • 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 relates generally to methods and compositions for treating bacterial infections, in particular infections of the respiratory tract, and for treating chronic obstructive pulmonary disease, using antagonists of the miRNA miR-328-3p.
  • Bacterial infections of the respiratory system are common, causing often serious and/or chronic health problems for sufferers as well as resulting in significant costs for health systems and economies worldwide.
  • Numerous bacterial species are known to cause respiratory tract infections, including Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenza.
  • Nontypeable Haemophilus influenzae is an important pathogen, particularly in children, causing or contributing to many diseases including pneumonia, bronchitis, otitis media, sinusitis, conjunctivitis, meningitis, septicaemia, and endocarditis.
  • NTHi Nontypeable Haemophilus influenzae
  • antibiotic resistant strains are becoming increasingly prevalent.
  • NTHi is also one of the most commonly isolated bacteria from individuals with chronic lung disease and is a major contributing factor to exacerbations and hospitalisations in those suffering from chronic obstructive pulmonary disease (COPD) and asthma.
  • COPD chronic obstructive pulmonary disease
  • NTHi infection macrophages and neutrophils are recruited to combat the infection. Host-pathogen interactions and cellular activation by the infecting bacteria can lead to increased cytokine secretion and phagocytosis by the immune cells to facilitate bacterial clearance.
  • MicroRNAs are small, single stranded, non-coding RNAs which regulate both mRNA degradation and translation, at least partially through their ability to bind to the 3' untranslated region (3'UTR) of target genes through base pairing with the 5'- end of the miRNA, via the so called seed sequence or seed region of the miRNA.
  • miRNAs themselves may be used to regulate the activity of target RNAs, opening the opportunity to develop miRNAs as therapeutics.
  • Mature miRNAs are derived from so-called pri-miRNAs that are transcribed from regions of non-coding DNA.
  • Pri-miRNAs usually containing several hundred nucleotides, are processed into stem-loop precursors (pre-miRNAs) of approximately 70 nucleotides by RNase III endonuclease.
  • Pre-miRNAs are actively transported into the cytoplasm where they are further processed into short RNA duplexes, typically of 21 -23 bp.
  • the functional miRNA strand dissociates from its complementary non-functional strand and locates within the RNA-induced-silencing-complex (RISC).
  • RISC RNA-induced-silencing-complex
  • RISC can directly load pre-miRNA hairpin structures.
  • the miRNA-RISC complex incompletely binds to its cognate mRNA target through a small region at the 5' end. miRNA-induced regulation of gene expression is then typically achieved by translational repression, either degrading proteins as they emerge from ribosomes or 'freezing' ribosomes, and/or promoting the movement of target mRNAs into sites of RNA destruction.
  • miRNAs are important in a number of developmental processes, for example in differentiation and maintenance of cellular identity in hematopoiesis, in establishing muscle phenotypes, in morphogenesis of epithelial tissues, organogenesis and in other metabolic processes. Additionally, specific miRNAs are increasingly being implicated in disease conditions, including cancers.
  • a first aspect of the present disclosure provides an immunostimulatory composition comprising at least one agent capable of inhibiting the expression and/or activity of miR- 328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the miR-328-3p is hsa_miR-328-3p and comprises the nucleotide sequence set forth in SEQ ID NO:l .
  • the miR-328-3p precursor may comprise the nucleotide sequence set forth in SEQ ID NO:2.
  • the agent is an antisense oligonucleotide specific for the miRNA.
  • the antisense oligonucleotide may comprise the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO;4.
  • the composition stimulates the clearance of a bacterial infection from a host.
  • the composition may stimulate phagocytosis of bacterial cells by host phagocytes.
  • the phagocytes may be macrophages or neutrophils.
  • the bacteria is nontypeable Haemophilus influenzae NTHi) or Escherichia coli.
  • a second aspect provides the use of an agent capable of inhibiting the expression and/or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU, as an immunostimulant.
  • a third aspect provides a method for treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and/or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the bacterial infection may be an infection of the respiratory tract.
  • the bacterial infection may be associated with an inflammatory or allergic airways disease in the subject.
  • the airways disease may be chronic obstructive pulmonary disease (COPD), asthma or cystic fibrosis.
  • COPD chronic obstructive pulmonary disease
  • the bacterial infection may be a nontypeable Haemophilus influenzae (NTHi) or Escherichia coli infection.
  • NHi nontypeable Haemophilus influenzae
  • Escherichia coli infection may be a nontypeable Haemophilus influenzae (NTHi) or Escherichia coli infection.
  • the miR-328-3p is hsa_miR-328-3p and comprises the nucleotide sequence set forth in SEQ ID NO:l .
  • the miR-328-3p precursor may comprise the nucleotide sequence set forth in SEQ ID NO:2.
  • the agent is an antisense oligonucleotide specific for the miRNA.
  • the antisense oligonucleotide may comprise the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.
  • Treating the bacterial infection may comprise promoting bacterial clearance.
  • Promoting bacterial clearance may comprise stimulating phagocytosis of bacterial cells by phagocytes, such as macrophages and neutrophils.
  • a fourth aspect of the present disclosure provides a method for promoting bacterial clearance from a subject, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the bacterial clearance may be from the respiratory tract of the subject.
  • the bacterial clearance may be from the lungs of the subject.
  • the bacterial clearance may be from other mucosal surfaces of the host including the genital tract and gastrointestinal system.
  • the bacteria is nontypeable Haemophilus influenzae (NTHi) or Escherichia coli.
  • a fifth aspect of the present disclosure provides a method for stimulating phagocytosis of bacteria by phagocytes, the method comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the bacteria is nontypeable Haemophilus influenzae (NTHi) or Escherichia coli.
  • a sixth aspect of the present disclosure provides a method for improving airway conductance in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • a seventh aspect of the present disclosure provides a method for reducing airway hyperresponsiveness in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • An eighth aspect of the present disclosure provides a method for treating an airways disease, or at least one symptom or exacerbation thereof, in a subject, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the airways disease may be, for example, COPD, asthma or cystic fibrosis.
  • a ninth aspect of the present disclosure provides a method for treating an immunodeficient subject, and/or improving innate immune cell functioning in an immunodeficient subject, the method comprising administering to the subject an effective amount of an agent capable of inhibiting the expression and or activity of miR-328-.3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • Figure l.miR-328-3p is down-regulated after NTHi infection, (a) NTHi clearance in lungs. Mice were infected i.t with 5 x 10 5 CPU of NTHi. Bacterial load in lungs determined by colony counts, (b) Total cells in BAL fluid following NTHi challenge, (c) Macrophages and (d) neutrophils numbers determined by differential cell counts, (e) Expression of miR-328-3p validated using Taqman PCR, normalized against sno-202 and expressed as fold change compared to PBS control group. Results are expressed as mean ⁇ SEM (* p ⁇ 0.05, **p ⁇ 0.01, *** pO.OOl ). CFU, colony forming units; BAL, bronchoalveolar lavage.
  • FIG. 3 Figure 2.miR-328-3p expression is regulated by p38 MAPK and knockdown of miR-328-3pw vitro increases bacterial clearance.
  • Primary lung macrophages were isolated from naive mouse lungs and pre-treated with antagomir-328 for 12 hours before infection with NTHi MOI 100.
  • Scrambled antagomir was used as a control, (a) miR-328-3p knockdown by antagomir-328 validated using Taqman PCR, normalized against sno-202 and expressed as fold change compared to scrambled control, (b) Numbers of extracellular bacteria in the supernatant were measured 8 hours post-infection by bacterial colony count, (c) Intracellular bacteria count was performed using the gentamicin exclusion assay to kill extracellular bacteria before cells were lysed to release intracellular bacteria, (d, e) Phagocytosis assay using heat-killed NTHi. Bacteria were labelled with CFSE and heat- killed before being added to macrophages for 1 hour.
  • Macrophage intracellular CFSE expression was analysed using (d) flow cytometry and (e) fluorescence microscopy where DAPI (blue) represents cellular DNA, Rhodamine (red) cell protein, and CFSE (green) heat-killed NTHi. (f) Confocal microscopy of cathepsin D expression 8 hours post NTHi infection.
  • DAPI blue
  • CFSE green
  • cathepsin D red
  • DHE Dihydroethidium
  • flow cytometry was used to monitor superoxide production in macrophages 8 hours following NTHi infection
  • Macrophages were pre-treated with 5 uM doramapimod for 30min to inhibit p38 activation prior to NTHi infection for 8hr and then miR-328-3p expression was measured using taqman PCR normalized against sno-202 and expressed as fold change compared to PBS control
  • miR-328-3p expression was measured using taqman PCR normalized against sno-202 and expressed as fold change compared to PBS control
  • CFU colony forming units
  • DAPI
  • CFSE Carboxyfluoresceinsuccinimidyl ester
  • CTSD cathepsin D
  • DMSO dimethyl sulfoxide Scr, scrambled control
  • Ant-328 antagomir-328.
  • Scrambled antagomir was used as control, (a) miR-328-3p knockdown by antagomir-328 was validated using Taqman PCR, normalized against sno-202 and expressed as fold change compared to scrambled control, (b) Extracellular bacteria in the supernatant was measured 1 hour postinfection by bacterial colony count, (c) Gentamicin exclusion assay was performed to kill extracellular bacteria before cells were lysed to release intracellular bacteria, (d) Phagocytosis assay using heat-killed NTHi. Bacteria were labelled with CFSE and heat- killed before being added to neutrophils for 1 hour.
  • mice were then adoptively transferred with macrophages or neutrophils (i.t.) followed by NTHi infection (i.t.) according to the timelines depicted.
  • Bacterial load in the lungs of mice adoptively transferred with (a) macrophages or (b) neutrophils was measured by bacterial colony counts, (c-e)
  • mice were first infected with NTHi for 6 hours before antagomir-328 or scrambled antagomir was administered to the lungs (i.t.) as a treatment.
  • FIG. 5 Effect of miR-328-3p on bacterial clearance in dexamethasone immune suppressed mice.
  • (a-f)Mice were treated with vehicle or dexamethasone (i.p) for 3 consecutive days before they were challenged with NTHi (i.t) for 18 hours according to the timelines depicted,
  • (d-f) Mice were treated with antagomir-328 or scrambled control at 6hours post NTHi challenge,
  • (a, d)miR-328-3p expression was determined using Taqman PCR, normalized against sno-202 and expressed as fold change compared to the vehicle and scrambled control groups,
  • (b, e) bacterial load in the lungs was measured by bacterial colony counts, and
  • (c, f) BAL fluid was collected to enumerate the total cellular infiltrate.
  • Results are expressed as mean ⁇ SEM (* p ⁇ 0.05, **p ⁇ 0.01, *** p ⁇ 0.001).CFU, colony forming units; BAL, bronchoalveolar lavage; Scr, scrambled control; Ant-328, antagomir- 328. CFU, colony forming units; BAL, bronchoalveolar lavage; Scr, scrambled control; Ant-328, antagomir-328;Dex, Dexamethasone.
  • FIG. 6 Inhibiting miR-328-3p improves bacterial clearance and airway function in chronically cigarette smoke-exposed mice. Mice were exposed to cigarette smoke daily for a period of 8 weeks in an established model of cigarette smoke-induced emphysematous lung changes.
  • mice were challenged with NTHi(i.t) for 6 hours and treated with antagomir-328 or scrambled control, (a) miR-328- 3p expression was determined using Taqman PCR, normalized against sno-202 and expressed as fold change compared to the air-exposed control group, (b) Bacterial load in the lungs at 18 hours post-infection was measured 'by bacterial colony count, (c) BAL fluid was collected to measure total cell infiltrates.
  • Airway function in terms of (d) compliance, and (e) elastance were measured using the flexivent system, (f) Mucous cell mucin-5ac mRNA expression was measured in the lungs using quantitative PCR, normalized against HPRT housekeeping gene and expressed as fold change compared to the scrambled control. Results are expressed as mean ⁇ SEM (* p ⁇ 0.05, **p ⁇ 0.01). CFU, colony forming units; BAL, bronchoalveolar lavage; Scr, scrambled control; Ant-328, antagomir-328.
  • FIG. 7 Inhibition of miR-328-3p in human monocyte-derived macrophages and neutrophils increases bacterial uptake.
  • Human monocytes and neutrophils were isolated from healthy adult blood and macrophages differentiated in vitro. Cells were pre-treated with antagomir-328 or a scrambled control. Human monocyte-derived macrophages were infected with NTHi for 8 hours at MOI 100.
  • Intracellular bacteria colony count was performed using gentamicin exclusion assay to kill extracellular bacteria before cells were lysed to release intracellular bacteria. Human neutrophils were infected with NTHi for 1 hour at MOI 10.
  • FIG. 8 Inhibition of miR-328-3p in vivo by antagomir-328 treatment improves OVA-induced airway hyperresponsiveness and reduces mucous production in a mouse model of asthma.
  • Mice were sensitized to ovalbumin (OVA) antigen (asthma group) or PBS (control group) on day 0 by intraperitoneal injection followed by challenge with OVA antigen on days 12, ⁇ 13, 14, and 15.
  • OVA ovalbumin
  • Mice were treated with either antagomir-328 or a scrambled control on days 12 and 14.
  • Airway resistance and (b) dynamic airway compliance were measured and calculated as a percentage change over baseline in response to increasing doses of inhaled methacholine.
  • FIG. 9 Knockdown of miR-328-3p in vitro increases E. coli clearance by mouse primary lung macrophages.
  • Primary lung macrophages were isolated from naive mice and pre-treated with antagomir-328 for 12 hours to knockdown miR-328-3p expression before infection with E. coli.
  • CFU of E. coli were determined in culture supernatant by bacterial colony counts.
  • CFU colony forming units; Scr, scrambled control; Ant-328, antagomir- 328.
  • the subject specification contains amino acid and nucleotide sequence information prepared using the programme Patentin Version 3.4, presented herein in a Sequence Listing. Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO: l represents the nucleotide sequence of mature hsa_miR-328-3p
  • SEQ ID NO:2 represents the nucleotide sequence of the precursor molecule of hsa_miR-328-3p.
  • SEQ ID NOs:3 and 4 depict the nucleotide sequence of an antisense oligonucleotide (antagomir) specific for hsa_miR-328-3p, while SEQ ID NO:5 shows the sequence of a scrambled (negative control) antagomir.
  • oligonucleotide refers to a single-stranded sequence of ribonucleotide or deoxyribonucleotide bases, known analogues of natural nucleotides, or mixtures thereof.
  • oligonucleotide comprises a nucleic-acid based molecule including DNA, RNA, PNA, LNA, UNA or any combination thereof.
  • An oligonucleotide that predominantly comprises ribonucleotide bases, natural or non-natural, may be referred to as an RNA oligonucleotide.
  • Oligonucleotides are typically short (for example less than 50 nucleotides in length) sequences that may be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences.
  • Antisense oligonucleotides are oligonucleotides complementary to a specific DNA or RNA sequence.
  • an antisense oligonucleotide is an RNA oligonucleotide complementary to a specific mRNA or miRNA.
  • the antisense oligonucleotide binds to and silences or represses, partially of fully, the activity of its complementary miRNA. Not all bases in an antisense oligonucleotide need be complementary to the 'target' or miRNA sequence; the oligonucleotide need only contain sufficient complementary bases to enable the oligonucleotide to recognise the target.
  • An oligonucleotide may also include additional bases.
  • the antisense oligonucleotide sequence may be an unmodified ribonucleotide sequence or may be chemically modified or conjugated by a variety of means as described herein.
  • polynucleotide refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof.
  • a "polynucleotide” comprises a nucleic-acid based molecule including DNA, RNA, PNA, LNA, UNA or any combination thereof.
  • the term includes reference to the specified sequence as well as to the sequence complimentary thereto, unless otherwise indicated.
  • Polynucleotides may be chemically modified by a variety of means known to those skilled in the art.
  • a "polynucleotide” comprises a nucleic-acid based molecule including DNA, RNA, PNA, LNA, UNA or any combination thereof.
  • nucleotide refers to a single nucleobase within the oligonucleotide or polynucleotide.
  • the nucleobase may be part of a DNA, RNA, INA, LNA, UNA(or combination of any two or more thereof) oligonucleotide or polynucleotide.
  • the nucleobase may be a universal base. Modified nucleobases are also contemplated by the present invention, as described hereinbelow.
  • the term "complementary" as used in refers to the ability of two single-stranded nucleotide sequences to base pair, typically according to the Watson-Crick base pairing rules, that is, between G and C and between A and T or U. In some embodiments, G also pairs to U and vice versa to form a so-called wobble base pair.
  • the base inosine (I) may be included within an oligonucleotide of the invention. I base pairs to A, C and U.
  • universal bases may be used. Universal bases can typically basepair to G, C, A, U and T. Often universal bases do not form hydrogen bonds with the opposing base on the other strand.
  • a complementary sequence refers to a contiguous sequence exclusively of Watson-Crick base pairs. For two nucleotide molecules to be complementary they need not display 100% complementarity across the base pairing regions, but rather there must be sufficient complementarity to enable base pairing to occur. Thus a degree of mismatching between the sequences may be tolerated and the sequences may still be complementary.
  • the term "capable of base pairing with” is used interchangeably with “complementary to”.
  • substitution refers to a nucleobase at a particular position within an oligonucleotide or polynucleotide having been substituted for another nucleobase.
  • the substitution may be, for example, because of the presence of a single nucleotide polymorphism in the target RNA.
  • substitution also encompasses deletions of nucleobases and additions of nucleobases.
  • blockmir refers to a steric blocking oligonucleotide that binds to an RNA target blocking the ability of one or more miRNA species from binding to, and affecting the activity of, said target. Blockmirs are constructed so as to be incapable of recruiting cellular RNAi machinery or RNase H. Blockmirs are described, for example, in WO 2008/061537 and WO 2012/069059, the disclosures of which are incorporated herein by reference. [0054] It will be understood that as used herein the term “expression” may refer to expression of a polypeptide or protein, or to expression of a polynucleotide or gene, depending on the context.
  • the polynucleotide may be coding or non-coding (e.g. miRNA). Expression of a polynucleotide may be determined, for example, by measuring the production of RNA transcript levels. Expression of a protein or polypeptide may be determined, for example, by immunoassay using an antibody(ies) that bind with the polypeptide.
  • the term "activity" as it pertains to a polynucleotide e.g. a DNA, mRNA or miRNA
  • protein or polypeptide means any one or more cellular function, action, effect or influence exerted by the polynucleotide, protein or polypeptide.
  • association with when used in the context of a disease or condition "associated with” bacterial infection means that the disease or condition may result from, result in, be characterised by, or otherwise associated with the bacterial infection.
  • association between the disease or condition and the infection may be direct or indirect and may be temporally separated.
  • airways disease refers to any respiratory or lung disease including chronic lung diseases.
  • airways disease may be regarded as synonymous with respiratory or lung disease and the terms may be used interchangeably herein.
  • the term "effective amount” includes within its meaning a non-toxic but sufficient amount or dose of an agent or compound to provide the desired effect.
  • the exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • treating refers to any and all uses that remedy a condition or symptoms, prevent the establishment of a condition or disease, or otherwise prevent, hinder, retard, or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever.
  • treating does not necessarily imply that a patient is treated until total recovery.
  • the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms.
  • methods of the present invention involve "treating" the disorder in terms of reducing or ameliorating the occurrence of a highly undesirable event associated with the disorder or an irreversible outcome of the progression of the disorder but may not of itself prevent the initial occurrence of the event or outcome. Accordingly, treatment includes amelioration of the symptoms of a particular disorder or preventing or otherwise reducing the risk of developing a particular disorder.
  • subject refers to mammals and includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals.
  • livestock animals eg. sheep, pigs, cattle, horses, donkeys
  • laboratory test animals eg. mice, rabbits, rats, guinea pigs
  • companion animals eg. dogs, cats
  • the present disclosure is predicated on the inventors' findings that the miRNA miR-328-3p is down regulated during NTHi infection, and further that by inhibiting miR- 328-3p bacterial clearance is enhanced via increased bacterial uptake by phagocytes.
  • Adoptive transfer of miR-328-depleted macrophages or neutrophils increased bacterial clearance in the lung, further supporting our in vitro observations and directly demonstrating that inhibition of miR-328 in these cells amplifies their ability to clear respiratory infections.
  • COPD chronic obstructive pulmonary disease
  • asthma including airway hyperresponsiveness, reduced airway conductance and mucous production
  • the inventors' findings exemplified herein demonstrate the therapeutic potential of miR-328-3p inhibition, as an immunostimulant to combat bacterial infections and treat diseases and conditions of the airways associated with bacterial infections, such as COPD and asthma.
  • embodiments of the present disclosure contemplate the treatment of immunodeficient subjects, and the improvement of innate immune cell functioning in such subjects, wherein an antagonist of miR-328-3p as described herein is administered to the subject.
  • kits for treating bacterial infections, for promoting bacterial clearance, and for stimulating phagocytosis of bacteria by phagocytes, such as macrophages and neutrophils comprising administering to a subject in need of treatment an effective amount of an agent capable of inhibiting the expression and/or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the present disclosure also provides compositions for carrying out such methods.
  • immunostimulatory compositions comprising an agent capable of inhibiting the expression and/or activity of miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • the present disclosure also provides compositions for carrying out such methods.
  • the present disclosure also provides compositions for carrying out such methods.
  • the immunodeficient subject may, for example, suffer from an immunodeficiency disease such as HIV, or may, for example, be a transplantation patient.
  • Embodiments of the present disclosure provide methods and compositions for treating bacterial infections, for promoting bacterial clearance, and for stimulating phagocytosis of bacteria by phagocytes in a host.
  • agents as described are typically administered to a subject in need of treatment, typically a subject having a bacterial infection.
  • the bacterial infection may be an infection of the respiratory tract, although the person skilled in the art will appreciate that the scope of the present disclosure is not limited thereto.
  • the bacteria causing, or associated with, the infection, and against which methods and compositions of the present disclosure may be employed may be Gram negative or Gram positive such as, for example, Haemophilus spp., Escherichia spp., Pseudomonas spp., Staphylococcus spp., Streptococcus spp., Neisseria spp., Mycoplasma spp., Klebsiella spp., Corynebacterium spp., Mycobacterium spp., Coxiella spp., Chlamydia spp., Enterobacter spp., Acinetobacter spp. and Enterococcus spp.
  • Non-limiting examples of bacterial species to which methods and compositions are applicable include Haemophilus influenzae, Escherchia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococous pneumoniae, Streptococcus pyogenes, Neisseria gonorrhoeae, Neisseria meningitis, Mycoplasma pneumoniae, Mycoplasma hominis, Klebsiella pneumoniae, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Moraxella catarhalis, Coxiella burnetii, Chlamydia pneumoniae, and Chlamydia trachomatis, in exemplary embodiments, the Haemophilus sp. is Haemophilus influenza, for example non- typeable Haemophilus influenza (NTHi).
  • NHi non- typeable Haemophilus influenza
  • the bacterial infection may be associated with an inflammatory or allergic airways disease such as COPD, asthma, allergic rhinitis, eosinophilic bronchitis, and cystic fibrosis.
  • the airways disease is COPD or asthma.
  • Methods and compositions of the present disclosure also provide for the treatment or alleviation of one or more symptoms or characteristics of airways disease such as COPD and asthma, for example including reducing airways hyperresponsiveness, improving airways conductance and/or reducing mucous production.
  • the miRNA comprising the seed sequence UGGCCCU is miR-328-3p.
  • the nucleotide sequence of mature human miR-328-3p (hsa_miR-328-3p) is provided in SEQ ID NO: 1, in which the seed sequence (or seed region)UGGCCCU is represented by positions 2 to 8. Additional sequence information for the miR-328-3p miRNA can be found at http://microrna.sanqer.ac.uk/sequehces/index.shtml.
  • variants of this miRNA include nucleotide sequences that are substantially similar to sequence of miR-328-3p.
  • a variant miRNA may comprise a sequence displaying at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
  • Embodiments of the present disclosure relate to the administration of agents capable of inhibiting or reducing the expression and/or activity of the miRNA miR-328-3p, a precursor or variant thereof or a miRNA comprising a seed region comprising the sequence UGGCCCU.
  • Suitable inhibitors may directly or indirectly effect miRNA activity or expression and may act at the level of the gene encoding the miRNA or the miRNA.
  • the inhibitor may effect a modulator or regulator of the expression or activity of a miRNA such as miR-328-3p, or a target of the miRNA.
  • the inhibitor may be a nucleic acid, proteinaceous or non-proteinaceous molecule.
  • the inhibitory agent may be referred to as an antagonist.
  • Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing the miRNA from carrying out its normal biological function.
  • the term "antagonist" is used hereinafter to refer to inhibitors of miRNA activity and/or of miRNA expression.
  • Suitable antagonists include inhibitory oligonucleotides and other antisense or inhibitory nucleic acids, antibodies (including monoclonal antibodies and antigen binding fragments),and organic and inorganic chemical compounds including small molecules.
  • the antagonist may be an antisense construct comprising a nucleotide sequence specific to miR-328-3p, or a portion thereof, wherein the antisense construct inhibits, at least partially, the expression or activity of the miRNA.
  • specific it is meant that the antisense construct is substantially specific for the miRNA, but not necessarily exclusively so. That is, while being specific for the miR-328-3p sequence, the antisense construct may also cross-hybridise with other sequences, such as other miRNA sufficient to inhibit expression.
  • the nucleotide sequence of an antisense construct may display less than 100% sequence identity with miR-328-3p and retain specificity thereto.
  • antisense constructs need not bind directly with the miRNA to which they are directed in order to effect the expression or activity of those miRNA. Binding of an antisense construct to its complementary cellular nucleotide sequence may interfere with transcription, RNA processing, transport, and/or stability of the miRNA to which it is specific.
  • An antisense molecule may comprise DNA, RNA, LNA, PNA nucleotides or nucleosides, or any combinations thereof.
  • Suitable antisense constructs for use in accordance with embodiments disclosed herein include, for example, antisense oligonucleotides, small interfering R As (siRNAs) and catalytic antisense nucleic acid constructs.
  • Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art. Typically oligonucleotides will be chemically synthesized on automated synthesizers.
  • an antisense oligonucleotide capable of binding to, and inhibiting the activity of miR-328-3p will typically bind to the seed region of the miRN A comprising the . sequence UGGCCCU.
  • the an exemplary sequence of a suitable antisense oligonucleotide specific for miR-328-3p has the nucleotide sequence shown in SEQ ID NO:3.
  • This exemplary oligonucleotide is 100% complementary to the mature sequence of human miR-328-3p, although those skilled in the art will readily appreciate that one or more base changes or substitutions may be made such that less than 100% complementarity exists whilst the oligonucleotide retains specificity for miR-328-3p and retains antagonistic activity against this miRNA.
  • Oligonucleotides for use in accordance with the present disclosure may comprise a variety of sequence and structural modifications, depending on the use and function of the oligonucleotide, as will be described further below. Those skilled in the art will appreciate that the sequence and structural modifications described herein are exemplary only, and the scope of the present invention should not be limited by reference to those modifications, but rather additional modifications known to those skilled in the art may also be employed provided the oligonucleotide retains the desired function or activity. For example, oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate miRNA target.
  • the oligonucleotide sequence may be modified by the addition of one or more phosphorothioate (for example phosphoromonothioate or phosphorodithioate) linkages between residues in the sequence, or the inclusion of one or morpholine rings into the backbone.
  • phosphorothioate for example phosphoromonothioate or phosphorodithioate
  • Alternative non-phosphate linkages between residues include phosphonate, hydroxlamine, hydroxylhydrazinyl, amide and carbamate linkages (see, for example, United States Patent Application Publication No.20060287260, Manoharan Li the disclosure of which is incorporated herein in its entirety), methylphosphonates, phosphorothiolates, phosphoramidates or boron derivatives.
  • nucleotide residues present in the oligonucleotide may be naturally occurring nucleotides or may be modified nucleotides.
  • modified nucleotides include 2'-0-methyl nucleotides, 2'-0- flouronucleotides, 2'-0-methoxyethylnucleotides, universal nucleobases such as 5-nitro- indole;LNA, UNA, PNA and INA nucleobases, 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA).
  • ribose sugar moiety that occurs naturally in ribonucleosides may be replaced, for example with a hexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group as described in United States Patent Application Publication No. 20060035254, Manoharan et al., the disclosure of which is incorporated herein in its entirety.
  • the oligonucleotide sequence may be conjugated to one or more suitable chemical moieties at one or both ends.
  • the oligonucleotide may be conjugated to cholesterol via a suitable linkage such as a hydroxyprohnol linkage at the 3' end.
  • Krutzfeldt J. et al., 2005, Nature 438:685-689, the disclosure of which is incorporated herein in its entirety.
  • Krutzfeldt et al. discloses the sequences of antagomirs comprising 2-O-methyl nucleotides, phosphorothioate linkages between residues at the 5' and 3' end, and a conjugated cholesterol moiety via a hydroxyprohnol linkage at the 3' end.
  • antagomir modified in the manner described in Krutzfeldt et al. as well as modifications or variations thereof.
  • the design of oligonucleotides or antagomirs for use in accordance with embodiments disclosed herein is well within the capabilities of those skilled in the art.
  • the sequence of a suitable antagomir to miR-328-3p is provided in SEQ ID NO:3.
  • the nucleotides present in the antagomir comprising the sequence of SEQ ID NO:3 may be 2'-0-methyl nucleoside phosphoroamiditeresidues (SEQ ID NO:4).
  • the antagomir may comprise the following sequence:
  • the antisense oligonucleotide comprises locked nucleic acid (LNA) residues, and is a so-called LNA inhibitor of miR-328-3p.
  • LNA locked nucleic acid
  • miRNA antagonists in the form of antisense oligonucleotides that bind to a target mRNA (typically the 3' UTR of a target mRNA) recognised by miR-328-3p rather than to the miRNA itself.
  • the antisense oligonucleotide is a biockmir, capable of blocking the regulatory activity of miR-328-3p at a particular miRNA binding site in target RNA, typically by blocking the activity of the RNAi machinery at a particular target RNA.
  • Biockmir oligonucleotides may do so by sequestering the target sequence (the miRNA binding site) of the target RNA, such that the RNAi machinery will not recognize the target sequence.
  • Modifications of interest may include those that increase the affinity of the oligonucleotide for complementary sequences, i.e.Mncreases the melting temperature of the oligonucleotide base paired to a complementary sequence, or increase the biostability of the oligonucleotide.
  • modifications include 2'-0-flouro, 2'-0-methyl, 2'-0- methoxyethyl groups.
  • LNA, UNA, PNA and ⁇ monomers may also be employed. For shorter oligonucleotides, typically a higher percentage of affinity increasing modifications are present. If the oligonucleotide is less than 12 or 10 nucleobases in length, it may be composed entirely of affinity increasing units.
  • the oligonucleotide binds to a region of a target molecule representing the complement of the seed sequence of miR-328-3p, i.e. the target site for miR-328-3p binding to the 3' UTR of a target RNA (the 'anti-seed' region).
  • oligonucleotides for use in accordance with the present disclosure may be of any suitable length depending on the precise function or use of the oligonucleotide. Typically, the oligonucleotides are between 8 and 25 bases in length. Even more typically, the oligonucleotides are between 10 and 20 bases in length.For strong binding to its target RNA, the length of an oligonucleotide may be increased. In some cases, delivery into cells may be improved may using shorter oligonucleotides. Further, in other cases, the position of the oligonucleotide respective to the seed or anti-seed sequence of the target may be adjusted.
  • the position of bases complementary to the seed or anti-seed region may be adjusted such that they are placed for example at the 5'end of the oligonucleotide, at the 3'end of the oligonucleotide or in or towards the middle of the oligonucleotide.
  • Oligonucleotides may be capable of activating RNase H.
  • RNase H cleaves the RNA part of a RNA-DNA duplex and the structural requirements for RNase H activation are well-known to the skilled addressee.
  • oligonucleotides of the invention may be capable of recruiting the cellular RNAi machinery and directing the RNAi machinery to the target RNA. This may result in cleavage of the target RNA or translational repression of the target RNA.
  • the oligonucleotides may neither recruit the RNAi machinery nor activate RNase H.
  • Phosphorothioate internucleotide linkages may connect the residues in an oligonucleotide to improve the biostability of the oligonucleotide. All linkages of the oligonucleotide may be phosphorothioate linkages. In another embodiment, the fraction of phosphorothioate linkages may be less than 95%, less than 90%, less than 85 %, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50 %, more than 95%, more than 90%, more than 85 %, more than 80%, more than 75%, more than 70%, more than 65%, more than 60% and more than 50 %.
  • RNA interference refers to a means of selective post-transcriptional gene silencing by destruction of specific RNA by small interfering RNA molecules (siRNA).
  • the siRNA is generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated.
  • Double-stranded RNA molecules may be synthesised in which one strand is identical to a specific region of the miRNA transcript and introduced directly.
  • corresponding dsDNA can be employed, which, once presented intracellular ⁇ is converted into dsRNA.
  • Inhibiting the activity or expression of miR-328-3p may also be achieved using catalytic antisense nucleic acid constructs, such as DNAzymes and ribozymes, which are capable of cleaving miRNA transcripts.
  • Ribozymes for example, are targeted to, and anneal with, a particular sequence by virtue of two regions of sequence complementarity to the target flanking the ribozyme catalytic site. After binding the ribozyme cleaves the target in a site-specific manner.
  • the design and testing of ribozymes which specifically recognise and cleave miRNA sequences can be achieved by techniques well known to those in the art (for example Lieber and Strauss, (1995) Mol. Cell. Biol. 15:540-551 , the disclosure of which is incorporated herein by reference).
  • Also contemplated as antagonists of miR-328-3p are antibodies and antigen binding fragments directed to a product of a target mRNA recognised by miR-328-3p or to a protein involved in the production of miR-328-3p.
  • the antibody may be, for example, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single- chain, chimeric, synthetic, recombinant, hybrid, mutated, or grafted.
  • the antibody may be an antibody fragment such as a Fab, F(ab') 2 , Fv, scFv, Fd, dAb, and other antibody fragments which retain an antigen-binding function.
  • Antigen-binding domains typically comprise an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • an antigen-binding domain may comprise only a VL region or a VH domain.
  • an Fd may comprise only of a VH domain but retains an antigen-binding function.
  • Antagonists of mi -328-3p described and contemplated herein may be linked to another molecule, such as a protein or peptide (e.g. albumin,), anti-inflammatory agent or immunomodulatory agent.
  • the antagonist may be linked by chemical cross-linking or by recombinant methods.
  • the antagonist may be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life.
  • the polymer may be, for instance polyethylene glycol, polypropylene glycol, or polyoxyalkylenes.
  • Agents capable of inhibiting the expression and/or activity of miRNA such as miR- 328-3p may be administered in accordance with the embodiments disclosed herein in the form of pharmaceutical compositions, which compositions may comprise one or more pharmaceutically acceptable carriers, excipients or diluents.
  • Such compositions may be administered in any convenient or suitable route such as by parenteral (e.g. subcutaneous, intraarterial, intravenous, intramuscular), oral (including buccal and sublingual), nasal or topical routes.
  • parenteral e.g. subcutaneous, intraarterial, intravenous, intramuscular
  • oral including buccal and sublingual
  • nasal or topical routes e.g. subcutaneous, intraarterial, intravenous, intramuscular
  • administration may be regional rather than systemic.
  • Regional administration provides the capability of delivering very high local concentrations of the agent to the required site and thus is suitable for achieving the desired therapeutic effect whilst avoiding exposure of other organs of the body to the compound and thereby potentially reducing side
  • the specific dose level of a composition for any particular individual will depend upon a variety of factors including, for example, the activity of the specific agent(s) employed, the age, body weight, general health and diet of the individual to be treated, the time of administration, rate of excretion, and combination with any other treatment or therapy. Single or multiple administrations can be carried out with dose levels and pattern being selected by the treating physician. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of agent may be administered per kilogram of body weight per day. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenylpolysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the formulation must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilisation.
  • dispersions are prepared by incorporating the various sterilised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active agents When the active agents are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Compositions or preparations may be prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active.
  • Tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oil of wintergreen, or
  • oligonucleotides may be incorporated into sustained-release preparations and formulations.
  • Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
  • the compositions in liposome form may contain stabilisers, preservatives, excipients and the like.
  • the preferred lipids are the phospholipids and the phosphatidylcholines (lecithins), both natural and synthetic.
  • the agents may also be administered in the form of microparticles.
  • biodegradable microparticles formed from polylactide (PLA), polylactide-co- glycolide (PLGA), and epsilon-caprolactone ( ⁇ -caprolactone) may be used.
  • oligonucleotide agents as disclosed herein may be administered to a subject in need thereof in a vector.
  • the vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion and foreign sequences and introduction into eukaryotic cells.
  • the vector may be an expression vector capable of directing the transcription of the DNA sequence of an antisense molecule.
  • Suitable viral expression vectors include for example epstein-barr virus-, bovine papilloma virus-, adenovirus- and adeno-associated virus-based vectors.
  • the vector may beepisomal.
  • the use of a suitable episomal vector provides a means of maintaining the antisense molecule in the required target cells in high copy number extra- chromosomally thereby eliminating potential effects of chromosomal integration.
  • the present disclosure contemplates combination therapies, wherein agents as described herein are coadministered with other suitable agents that may facilitate the desired therapeutic outcome.
  • agents such as antibiotics
  • coadministered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the agents. Administration may be in any order.
  • mice [00106J Specific pathogen free adult male BALB/c mice were used in all experiments. Mice were obtained from the University of Newcastle Central Animal House. The animal protocols used in this study were conducted in accordance with guidelines of The University of Newcastle Animal Care and Ethics Committee. All human studies were conducted in accordance with the guideline of the Newcastle Human Research Ethics Committee.
  • Non-typeable Haemophilus Influenzae biotype II (NTHi 289), was kindly provided by A/Prof Margaret Dunkley (Hunter Immunology Ltd, Newcastle, Australia). Bacteria were prepared prior to infection by growing overnight on chocolate agar plates (Oxoid, Australia) at 37 °C in an atmosphere of 5% C0 2 .
  • mice were anaesthetised (12.5 mg/kg Alfaxan, Jurox, NSW, Australia) intravenously via the tail vein and innoculated intratracheally (i.t) using a cathether (Terumo, Sureflo Hospital Supplies of Australia) with 5xl 0 5 or 5xl 0 6 CFU of live NTHi and both BAL fluid and lung homogenate (homogenised in 1 ml sterile PBS (Gibco, Invitrogen, New Zealand)) were used to determine bacterial growth. Serial dilutions were loaded onto chocolate agar plates (Oxoid, Australia) and incubated overnight at 37 °C in an atmosphere of 5% C0 2 . After 16 hours, bacterial colonies for NTHi were counted.
  • Lung tissue from nai ' ve mice was mashed through a 70 ⁇ cell strainer before being layered on Histopaque 1083 and centrifuged (3,000rpm, 30min, 20°C).
  • the interface layer containing mononuclear cells was collected and allowed to adhere to a culture plate for 3 hr at a concentration of 1 x 10 6 cells before the non-adherent cells were removed.
  • Neutrophils were extracted from bone marrow of naive mice using a 3 layer Percoll gradient and magnetic bead separation. Briefly, bone marrow cells were isolated from femurs and tibias from both hind legs by flushing the bone with HBSS-EDTA solution. Erythrocytes were lysed with lysis buffer. Cells were then layered on a three- layer Percoll gradient of 78%, 69%, and 52% Percoll and centrifuged (l,500g, 20 min, 20°C) at the slowest deceleration.
  • Cells from the 69%/78% interface were collected and neutrophils further purified by negative immunornagnetic separation using labelling with anti-CD4, -CD8, -CD 1 1c, -CD49b, -CD 1 17, -B220, and -F4/80 purified antibodies (BD Biosciences, USA).
  • the labelled cells were then depleted through magnetic separation by first incubating with BD IMAG streptavidin particles plus-DM (BD Biosciences) according to manufacturer's protocol. The enriched fraction containing > 96% neutrophils was utilised.
  • RNA and miRNA quantitative RT-PCR were challenged with either NTHi or PBS as control and were sacrificed at 24 h post-challenge.
  • Total RNA was extracted from the airways using the Ambionmir VANA kit according to the manufacturer's protocol. RNA quality was determined using anbioanalyzer (Agilent). miRNA microarray analysis was performed with mouse miRNA microarray kit release 12.0 (Agilent) according to the manufacturer's protocol. Analysis of microarray data was conducted using Genespring GX software version 11.0. mRNA and miRNA quantitative RT-PCR
  • m represents 2 , -OMe-modified nucleoside phosphoramidites, "*” represents phosphorothioate linkages, and "-Choi” is hydroxyprolinol-linked cholesterol to allow permeation of cell membranes.
  • mice were treated in vivo with 50 ⁇ g of antagomir intratracheally, and macrophages and neutrophils were treated with antagomir at 5( ⁇ g/ml 12 hours before assays in vitro.
  • Macrophages were detached in citric saline (0.135 M CL, 0.015 M Na citrate) while neutrophils were removed by rinsing. Cells were spun down and resuspended in FACS buffer (2% FCS in PBS) before incubation with 10 ⁇ dihydroethidium (DHE) for 30min at 37 °C. Fluorescence intensity was measured by flow cytometry with 488nm excitation and 610nm emission.
  • NTHi was labelled with ⁇ CFSE (Molecular Probes) (before heat-killing) for 10 min at 37°C. Staining was quenched with fetal calf serum (FCS) for 1 min, and bacteria washed, resuspended in PBS, and incubated with cells for 1 hour. Cells were harvested as above for flow cytometry. Numbers of cells positive for CFSE and mean fluorescence intensity were measured using flow cytometry. To visualize the uptake of heat-killed NTHi, macrophages or neutrophils were initially adhered to coverslips.
  • FCS fetal calf serum
  • Macrophage and neutrophils were pre-treated with either scrambled control, antagomir to miRNA-328, or non-treated and were harvested and labelled with 5 ⁇ of CFSE (Molecular Probes).
  • a quantity of 5 x 10 5 CFSE labelled macrophages or neutrophils were administered to the lung of mice intratracheally in 40 ⁇ volume. 24 hours after macrophage instillation and 2hr after neutrophils instillation, mice were challenged with NTHi. Mice adoptively transferred with macrophages were sacrifice 12 h post infection while mice adoptively transferred with neutrophils were sacrificed 6 h post infection. Cytokine analysis
  • TNF-a and IL-6 were measured in the supernatant of BAL, lung homogenate, and in vitro cell culture supernatants using ELISA kits (ebioscience) according to manufacturer's instructions.
  • Macrophages were pre-treated with 5 ⁇ doramapimod (LC lab) for 30 min to inhibit p38 MAPK activation, prior to NTHi challenge for 8hr at MOI 10.
  • DMSO was used as a vehicle control.
  • the cells were lysed and the amount of protein was determined with BCA protein assay (Thermo Scientific). Protein samples (20 ⁇ g) were resolved on SDS electrophoresis gels, transferred on PVDF membrane by standard procedures and blotted for the protein of interest using an antibody to phosphorylated p38 (Abeam), ⁇ -actin expression were used for normalization. Bound antibodies were detected using chemiluminescent substrate (Bio- rad).
  • PBMCs were isolated by Ficoll centrigugation (GE Healthcare) and cells were adhered to cultures plates for 3 hours at 5xl0 6 cells/ml.. after which non-adherent cells were gently removed.
  • Adherent cells were cultured in 50ng/ml recombinant human M-CSF (Peprotech) to induce macrophage differentiation, and fresh media with M-CSF was replaced on day 3 and day 6 of culture.
  • Monocyte-derived macrophages were used on day 7. For neutrophil isolation the remaining Ficoll layer was removed without disturbing the layer of neutrophils/RBCs at the bottom.
  • the thin white cell layer of neutrophils above the RBC pellet was collected and resuspended in an equal volume of HBSS and dextran/saline solution (5% dextran T500 in 0.9% NaCl at room temperature), before being incubated in an upright position for 20min at room temperature.
  • the layer of neutrophils above the sedimented RBC layer was aspirated and remaining RBCs lysed with lysis buffer. Cells were resuspended in culture media ready for assays.
  • mice were exposed to cigarette smoke (in the form of 12 complete cigarettes) in a closed chamber twice a day, 5 days per week for a duration of 8 weeks as described in Beckett et al. (2013).
  • mice were injected intraperitoneally (i.p.) with 3mg/kg of dexamethasone (DEX) for 3 consecutive days before they were inoculated with (5 x 10 s CFU / mouse) with NTHi.
  • DEX dexamethasone
  • Example 1 -miRNA expression in the lung following NTHi infection
  • mice were infected tntratracheally (i.t.) with low dose (5 x 10 5 CFU) NTHi. NTHi reached peak bacterial loads between 6-12 hours and was cleared rapidly thereafter to the point where it was only slightly above baseline levels by 24 hours (Figure la).
  • Total cells ( Figure lb) and neutrophils (Figure Id) in the bronchoalveolar lavage (BAL) fluids increased 6 hours post NTHi infection while macrophages remained the same ( Figure lc).
  • miRNA-328-3p as a candidate miRNA as the baseline expression of this miRNA was among the highest, however uniquely it was also down-regulated rather than up-regulated or unchanged following NTHi infection. Additionally, the role of miR-328-3p in pathogen immunity, inflammation, and chronic inflammatory disorders , remains uncharacterised. The inventors validated the miRNA array chip expression of miR-328-3p using Taqman PCR and observed an approximately 2-fold reduction in expression (Figure le).
  • ROS reactive oxygen species
  • the inventors then determined whether other miRNAs that were also identified as having increased or decreased expression in the lungs of mice following NTHi infection, could also play a role in bacterial clearance.
  • Antagomir-mediated inhibition of miR-21-3p and miR-223 (increased expression following bacterial infection), miR-376c (decreased expression), and miR-21 (control, no change in expression) had no effect on NTHi clearance by neutrophils in vitro (data not shown).
  • mice that received antagomir-328 treated macrophages or neutrophils showed significantly improved clearance of NTHi from the lungs compared to scrambled control treatment ( Figure 4a and b).
  • the BAL cell counts showed no difference in inflammatory cell infiltrates between mice receiving antagomir-328 treated cells and scrambled control treated cells (data not shown).
  • Example 3 Inhibition of mir-328 in the lungs overcomes corticosteroid-mediated immune suppression to clear bacteria
  • NTHi is one of the most common bacteria isolated from the airways of both stable COPD patients and those undergoing acute exacerbations (Monso et al, 1995). Therefore, the inventors investigated if miR-328-3p affects bacterial clearance in a COPD/emphysema model. Using an established model of cigarette smoke-induced emphysema mice were exposed to cigarette smoke for 8 weeks and then were infected with NTHi followed by antagomir-328(SEQ ID NO:4) treatment. Knockdown of miR-328-3p expression using antagomir-328 was validated in this model (Figure 6a).
  • MiR-328-3phas the same sequence in humans as in mice.
  • Neutrophils were purified from blood of healthy adults, and macrophages were differentiated in culture from monocytes derived from the PBMC fraction.
  • Antagomir-328 (SEQ ID NO:4) achieved effective inhibition of miR-328-3p expression in both human macrophages and neutrophils (data not shown). Similar to the results observed in mice, inhibition of miR-328-3p significantly increased bacterial phagocytosis by both human macrophages (Figure 7a) and neutrophils ( Figure 7b) in vitro.
  • Ovalbumin (OVA)-induced allergic asthma is a widely used model to reproduce the mucous hypersecretion, pulmonary inflammation, and airway hyper-responsiveness (AHR) that are classic pathologic features of asthma.
  • AHR airway hyper-responsiveness
  • mice were sensitized with an intraperitoneal injection of OVA on day 0 and then rechallenged with 50 ⁇ 1 OVA on days 12, 13, 14, and 15, delivered intranasally.
  • 5C ⁇ g antagomir-328 (SEQ ID NO:4) or scrambled antagomir (control; SEQ ID NO:5) was administered on days 12 and 14 during the asthma challenge phase.
  • AHR was measured and lung tissue was stained to identify mucous producing cells.
  • AHR was assessed invasively in anesthetized mice by measurement of airway resistance and dynamic compliance in response to increasing doses of methacholine. Percentage increase over baseline (saline) in response to nebulized methacholine was calculated. Lung tissue was stained with Periodic acid-Schiff and mucous producing cells were identified by counting ten high-powered fields for each lung section.
  • the inventors also investigated the effect of miR-328-3p inhibition on the clearance of other bacteria including Escherichia coli from mouse primary lung macrophages.
  • Macrophages isolated from naive mouse lungs were pre-treated with antagomir-328 (SEQ ID NO:4) for 12 h to inhibit miR-328-3p expression before being infected with E. coli at a multiplicity of infection of 10: 1.
  • Scrambled antagomir SEQ ID NO:5 was used as a control.
  • Extracellular bacteria in the supernatant were measured 8 hours post-infection by bacterial colony count.
  • Macrophages treated with antagomir-328 demonstrated a substantially increased clearance of£. coli compared to the scrambled control (Figure 9).

Abstract

La présente invention concerne des compositions immunostimulatrices comprenant au moins un agent apte à inhiber l'expression et/ou l'activité de miR-328-3p, son précurseur ou son variant, ou un miARN comprenant une région de germes qui comporte la séquence UGGCCCU. L'invention porte en outre sur des utilisations desdites compositions notamment dans le traitement d'infections bactériennes, la promotion de la clairance bactérienne, et le traitement de maladies des voies aériennes et de l'immunodéficience.
PCT/AU2013/001421 2012-12-06 2013-12-06 Utilisation d'antagonistes de miarn pour la modulation du système immunitaire, traitement d'infections bactériennes et traitement de maladies des voies aériennes WO2014085866A1 (fr)

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