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WO2007039759A1 - Association du gene fbx011 a l'otite moyenne - Google Patents

Association du gene fbx011 a l'otite moyenne Download PDF

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Publication number
WO2007039759A1
WO2007039759A1 PCT/GB2006/003731 GB2006003731W WO2007039759A1 WO 2007039759 A1 WO2007039759 A1 WO 2007039759A1 GB 2006003731 W GB2006003731 W GB 2006003731W WO 2007039759 A1 WO2007039759 A1 WO 2007039759A1
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Prior art keywords
polypeptide
fbxoil
amount
fbxoi
fbxol
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Stephen David Mcleod Brown
Rachel Elizabeth Hardisty-Hughes
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Medical Research Council
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/14—Disorders of ear, nose or throat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention relates to diagnosis and treatment of otitis media (OM) 5 genes and polypeptides which are involved in OM and methods for obtaining and using animal models for OM.
  • OM otitis media
  • Otitis Media inflammation of the middle ear lining
  • OM Otitis Media
  • Evidence from studies of the human population demonstrates that there is a significant genetic component to the development of OM (Kvaerner et al Ann Otol Rhinol Laryngol. 1997 Aug;106(8):624-32; Casselbrant et al. JAMA. 1999 Dec 8;282(22):2125-30; Hardisty et al. J Assoc Res Otolaryngol. 2003 Jun;4(2):130-8; Casselbrant & Mandel Curr Opin Allergy Clin Immunol. 2005 Feb;5(l):l-4), yet little is known about the underlying genetic pathways involved.
  • Acute OM is an infection that involves the middle ear.
  • the tympanic membrane becomes inflamed and opaque. Blood vessels to the area dilate. Fluid accumulates in the middle ear space. It is usually associated with infection by viruses or bacteria: In a 1990 surveillance by the Centers for Disease Control and Prevention otitis media was the most common principal illness diagnosis in children 2-10 years of age.
  • the most common causes of acute OM are Streptococcus pneumonia, Haemophilus influenzae and Moraxella catarrhalis. Less commonly, Mcyoplasma pneumoniae, Streptococcus pyogenes, Staphylococcus aureus along with other bacteria and viruses may be involved.
  • Chronic OM with effusion is a persistent inflammation and accumulation of sticky fluid, or effusion, in the middle ear.
  • Chronic OM may develop within weeks of an acute episode of middle ear infection, but in many cases the cause is unknown. It occurs when the eustachian tube becomes blocked repeatedly (or remains blocked for long periods) due to allergies, multiple infections, ear trauma, or swelling of the adenoids.
  • Ciprodex contains a mixture of antibiotic and anti- inflammatory (dexamethasone 0.1% and ciprofloxacin 0.3%) active components.
  • Wyeth's S. pneumoniae vaccine Prevnar is indicated for active immunization of infants and toddlers against OM caused by serotypes included in the vaccine and in some studies has been shown to reduce the number of physician visits for acute OM.
  • Novel antibiotic therapies with an indication for acute OM include Arpida's AR-709 (pre-clinical) and Enanta's EP-013420 (Phase I).
  • Vaccines in the pipeline with indications for OM include ID Biomedical's SPD-703 directed against pneumoniae species (Phase I) and Apovia's OtiVax (pre-clinical) directed against Haemophilus influenzae and Moraxella catarrhalis. These bacteria are responsible for about 40%-50% of acute OM infections hi children.
  • Chronic OM is often the target of surgical intervention (by plastic tympanostomy ventilation tube (grommet) insertion to relieve middle ear pressure); 5-600,000 OM-related middle ear surgeries are performed per annum in the US alone. Chronic OM remains the most common cause of surgery in children in the developed world with enormous heath care costs. In addition to being expensive, surgery is also unpopular and recent concerns regarding its effectiveness have been raised, hi a study by Paradise et at. N Engl J Med.
  • OM therapies There are few products in development as non-surgical chronic OM therapies. They include Arriva's protease inhibitor therapies Alpha- 1 antitrypsin (rAAT) and Ilomastat (both pre-clinical). High levels of the schooleases neutrophil elastatse (NE) and the matrix metalloproteinases (MMPs) are found in the exudates of adult and pediatric patients with middle ear disease. The excessive protease activity of NE and MMPS could be neutralised using Arriva's rAAT and Ilomastat, respectively.
  • rAAT Alpha- 1 antitrypsin
  • Ilomastat both pre-clinical
  • NE neutrophil elastatse
  • MMPs matrix metalloproteinases
  • OctoPlus' (phase I/IIa) OP-145 is a recombinant analogue of the bactericidal/permeability-increasing (BPI) protein capable of inhibiting endotoxin contained within a proprietary drug delivery technology for the treatment of mucosal infections such as chronic OM.
  • BPI bactericidal/permeability-increasing
  • the middle ear is an accessible organ for the trial and application of new therapeutics.
  • Novel small molecule or peptide therapeutics or gene therapy therapeutics could be delivered directly (ototopically) as well as systemically to the middle ear.
  • Ototopic delivery may have the advantage of restricting the vicinity in which the drug is deposited thereby potentially increasing potency and minimising undesirable side effects.
  • utilising tympanostomy tubes as an opening to the middle ear, or placement of a biodegradable support matrix containing the therapeutically active component in the middle ear e.g. Goycoolea et al. Acta Otolaryngol Suppl.
  • the Jeff mutant mouse (Hardisty et al. 2003) is a mouse model demonstrating chronic suppurative OM in the absence of any other inflammatory pathology; this is typical of the disease in the human population.
  • the inventors have now cloned the mutant gene underlying the Jeff mutation, identifying a component of the genetic pathway that influences development of OM.
  • the mutant gene in Jeff is an F-box gene (Fbxoll), a component of the SCF E3 ligase complex involved in ubiquitination.
  • the F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction.
  • F-box proteins target substrate polypeptides for ubiquitination and subsequent protein degradation.
  • Recent reviews of protein ubiquitination include Robinson and Ardley Curr Protein Pept Sci. 2004 Jun;5(3):163-76.and J Cell Sci. 2004 Oct 15;117(Pt 22):5191-4. and Swinney, Drug Discov Today. 2001 Mar l;6(5):244-250..
  • the SCF ubiquitin ligase is composed of a modular E3 core containing CULl and RBXl (also called ROCl) and a substrate specificity module composed of SKPl and a member of the F-box family of proteins.
  • F-box proteins carry an F-box for interaction with the SKPl protein of the SCF complex. Additionally, each F-box protein carries a unique set of motifs that is required for specifically binding the relevant cargo for ubiquitination.
  • Fbxol l contains two CASH domains frequently found in carbohydrate-binding proteins.
  • F-box proteins there are 68 F-box proteins in humans and clear mouse orthologs exist for all but one. These proteins fall into three major classes, depending on the types of substrate interaction domains identified in addition to the F-box motif. The two largest classes of interaction domains are WD40 repeats [W] and leucine-rich repeats [L]. A third generic class of F-box proteins contains various other types of protein interaction domains or no recognizable domains [O refers to "other" domains]. These classes of F-box proteins are designated FBXWs 5 FBXLs and FBXOs, respectively, followed by a numerical identifier. For a review, see Jin et al Genes Dev. 2004 Nov l;18(21):2573-80.
  • FBXOlO and FBXOIl Two F-box proteins (FBXOlO and FBXOIl) contain the CASH domain frequently found in carbohydrate-binding proteins and hydrolases. Both D. melanogaster and C.elegans contain possible orthologs of FBXOlO and FBXOIl.
  • E3 ligases including F-box proteins and their related pathways may be attractive candidates for drug discovery because they play crucial roles in many important signalling pathways.
  • Specific agonists or antagonist of E3 ligase components may be appropriate therapies for some disease.
  • E3 ligases are suggested as good biological targets in the development of anticancer therapeutics.
  • E3s have established roles in cell cycle and apoptosis.
  • Millenium Pharmaceuticals' PS-341 Velcade (bortezomib), a proteasome inhibitor, has been approved by the FDA for the treatment of relapsed and refractory multiple myeloma.
  • E3s have been implicated in neurodegenerative disease and viral infections, amongst others.
  • Fbxoll was first described in Le Poole et al. Pigment Cell Res. 2001 Dec;14(6):475-84 as a gene which is down-regulated in vitiligo. Vitiligo is a pigmentation disorder in which melanocytes in the skin and mucous membranes are destroyed. Fbxoll has subsequently been identified as an F-box gene by homology, Fbxoll function was not previously known and no further references to Fbxoll function appear in the literature to date. Moreover, until the present invention FBXOl 1 was not thought to be involved in OM.
  • a first aspect of the invention provides a method for assessing a patient's risk of developing otitis media (OM); or progression of OM; or for assisting in the diagnosis of OM, comprising the steps of:
  • the amount of functional FXBOl 1 polypeptide is considered to be associated with the development or progression of OM, for example in the development of chronic OM or the progression of acute OM to chronic OM. Therefore, the amount and/or function of FBXOl 1 polypeptide, or determining the amount of nucleic acid encoding FBXOI l, or determining the patient's genotype of FBXOl 1 (or a combination thereof) can be a key indicator for assessing the risk for a patient of developing otitis media (OM) or progression of OM, for example the risk of development of chronic OM or of progression of acute OM to chronic OM; or can be useful in diagnosing OM. Such a method may include determining whether there is any change in the function of the FXBOl 1 polypeptide. The method of this aspect of the invention may be useful in the diagnosis of OM, for example chronic OM, or as a basis for genetic counseling.
  • OM otitis media
  • Such a method may include determining whether there
  • the step of determining the amount of FBXOI l polypeptide may be performed using any of a number of different methods.
  • Assaying the amount of FBXOI l polypeptide in a biological sample can be performed using any art-known method.
  • Preferred for assaying FBXOIl polypeptide levels in a biological sample are antibody-based techniques.
  • FBXOIl polypeptide expression can be studied with classical immunohistological methods.
  • the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies.
  • an immunohistological staining of tissue section for pathological examination is obtained. See, for example, Example 1.
  • Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of FBXOI l polypeptide for Western-blot or dot/slot assay (Jalkanen, M., et al. 5 J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al, J. Cell. Biol. 105:3087-3096 (1987)).
  • quantitation of FBXOl 1 polypeptide can be accomplished using isolated FBXOl 1 polypeptide as a standard. This technique can also be applied to animal fluids.
  • a molar concentration of FBXOIl polypeptide will aid to set standard values of FBXOIl polypeptide content for different animal fluids, like serum, plasma, urine, spinal fluid, etc.
  • the normal appearance of FBXOI l polypeptide amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject.
  • An example of a tissue that may be tested include skin.
  • FBXOI l polypeptide-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify the FBXOI l polypeptide.
  • the amount of FBXOIl polypeptide present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • two distinct specific monoclonal antibodies can be used to detect FBXOl 1 polypeptide in a body fluid.
  • one of the antibodies is used as the immunoadsorbent and the other as the enzyme-labelled probe.
  • FBXOIl 1 polypeptide can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of FBXOIl polypeptide include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject.
  • Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labelling of nutrients for the relevant hybridoma.
  • FBXOIl polypeptide-specific antibodies for use in the screening methods of the present invention can be raised against the intact FBXOI l polypeptide or an antigenic polypeptide fragment thereof, which may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to FBXOIl polypeptide.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (WaM et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the step of determining the function of FBXOl 1 polypeptide may be performed using a number of different methods known to those skilled in the art.
  • the substrate ubiquitinylation or autoubiquitinylation activity of the SCF E3 ligase complex containing the FBXOl 1 (for example immunoprecipitated from a patient sample using antibodies directed to FBXOl 1) may be assessed.
  • FBXOl 1 substrates could be identified by an in vitro pull-down assay, a well known technique to those skilled in the art, to determine physical interaction between the enzyme and its substrate.
  • FBXOI l -mediated ubiquitinylation of its substrate(s) could then be measured, for example, by using the method described by Yabuki et al. Comb Chem High Throughput Screen. 1999 Oct;2(5):279-87. where homogeneous time-resolved fluorescence is used to monitor poly- ⁇ biquitination of wild-type p53.
  • the step of determining the amount of nucleic acid encoding FBXOIl may be performed using a number of different methods.
  • RNA encoding the FBXOIl polypeptide may be assayed using the RT-PCR method described in Makino et al., Technique 2:295-301 (1990).
  • the radioactivities of the "amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA. Briefly, this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase.
  • RNA After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers.
  • a labeled dNTP can be included in the PCR reaction mixture.
  • PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of W the appropriate bands (corresponding to the rnRNA encoding the FBXOI l polypeptide) is quantified using an imaging analyzer.
  • RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art.
  • RNA levels can be measured using any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as will be well known to those skilled in the art.
  • Alternative techniques by which to measure mRNA levels include incorporation of SybrGreen or other fluorophores into primers or probes as part of RealTime PCR experiments.
  • RNA encoding the FBXOl 1 polypeptide can also be assayed using northern blotting, a method well known to those skilled in the art and described further in Sambrook et al.., Molecular Cloning. A laboratory manual. 1989. Cold Spring Harbour publications.
  • the step of determining the patient's genotype for the FBXOI l gene may be performed using a number of different methods.
  • genomic rearrangements can lead to an increase in the copy number of gene(s) encoding FBXOIl polypeptide, i.e. nucleic acids encoding said polypeptide.
  • Methods of determining gene copy number include Southern blotting (essentially as performed as set out in Sambrook et al. (1989). Molecular cloning, a laboratory manual, 2 nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York) or quantitative PCR.
  • Methods of determining the person's genotype for the FBXOIl gene include determining whether the person has one or more mutation(s) in the gene, or complete absence of the gene(s) encoding FBXOl 1 polypeptide, leading to altered expression of the ⁇ olypeptide(s) or expression of functionally inactive or functionally altered versions of the polypeptide(s).
  • gene the inventors include the coding region and the controlling region, e.g. the promoter, of the gene.
  • Such genetic assay methods include the standard techniques of restriction fragment length polymorphism assays and PCR-based assays, as well as DNA sequencing.
  • the assay may involve any suitable method for identifying such polymorphisms, such as: sequencing of the DNA at one or more of the relevant positions; differential hybridisation of an oligonucleotide probe designed to hybridise at the relevant positions of either the wild-type or mutant sequence; denaturing gel electrophoresis following digestion with an appropriate restriction enzyme, preferably following amplification of the relevant DNA regions; Sl nuclease sequence analysis; non- denaturing gel electrophoresis, preferably following amplification of the relevant DNA regions; conventional RFLP (restriction fragment length polymorphism) assays; selective DNA amplification using oligonucleotides which are matched for the wild-type sequence and unmatched for the mutant sequence or vice versa; or the selective introduction of a restriction site using a PCR (or similar) primer matched for the wild-type or mutant genotype, followed by a restriction digest.
  • any suitable method for identifying such polymorphisms such as: sequencing of the DNA at one or more of the relevant positions;
  • the assay may be indirect, ie capable of detecting a mutation at another position or gene which is known to be linked to one or more of the mutant positions.
  • the probes and primers may be fragments of DNA isolated from nature or may be synthetic. The methods used to determine genotype(s) are well known to those skilled in the art.
  • the method may comprise the step of determining whether or not the patient has one or more alleles of the FBXOl 1 gene in which there is a mutation in the region comprising the CASH domains, ie between amino acids 334 to 753 of FXBOI l, preferably between amino acids 334 to 470, 471 to 624 (region separating the two CASH domains) or 624 to 753.
  • the method may comprise the step of determining whether or not the patient has one or more alleles of the FBXOl 1 gene in which there is a mutation at position Q491, for example a mutation to a non-conservative substitution, for example to Leucine.
  • the method may comprise the step of determining whether or not the patient has one or more alleles of the FBXOIl gene in which there is a mutation at position S244, for example a mutation to a non-conservative substitution, for example to Leucine.
  • the method may be performed on any convenient tissue sample, as will be well known to those skilled in the art.
  • a "reference sample” i.e. a sample of protein or nucleic acid taken from a patient who does not have OM.
  • the patient is considered to be at risk of developing OM.
  • test may conveniently be performed immediately after birth or as part of post- natal screening, for example within six months of birth. Alternatively, tests may be performed at a later stage (for example up to about five years of age), for example if hearing difficulties are noted or if either acute or chronic OM is diagnosed or suspected.
  • a further aspect of the invention provides the use of an agent which is capable of being used in determining one or more of: (a) the amount and/or function of FBXOI l polypeptide; (b) determining the amount of nucleic acid encoding FBXOIl; and (c) determining a patient's genotype of FBXOIl, in the manufacture of a reagent for assessing a patient's risk of developing otitis media (OM); or progression of OM; or for assisting in the diagnosis of OM.
  • an agent which is capable of being used in determining one or more of: (a) the amount and/or function of FBXOI l polypeptide; (b) determining the amount of nucleic acid encoding FBXOIl; and (c) determining a patient's genotype of FBXOIl, in the manufacture of a reagent for assessing a patient's risk of developing otitis media (OM); or progression of OM; or for
  • determining the amount and/or function of FBXOI l polypeptide and/or determining the amount of nucleic acid encoding FBXOI l and/or determining the patient's genotype of FBXOI l can be a key indicator for assessing the risk of a patient developing otitis media (OM) or in progression of OM, or diagnosing OM.
  • OM otitis media
  • agents that can be used in this aspect of the invention to determine the amount and/or function FBXOIl polypeptide include antibodies or peptide or compounds that can bind to said polypeptide. Such agents may be able to selectively bind polypeptide having the amino acid sequence of FBXO 11 in which the residue corresponding to Q491 is mutated, for example to L; and/or in which the residue corresponding to S244 is mutated, for example to L. Furthermore, as set out above in relation to the diagnostic method of the invention, agents that may be used to assess the function of FBXOIl polypeptide include agents useful in measuring ubiquitinylation.
  • Agents that can be used in this aspect of the invention to determine the amount of nucleic acid encoding FBXOI l polypeptide include nucleic acid molecules which hybridise preferably selectively to the nucleic acid encoding FBXOI l, such as primers, oligonucleotides, or other nucleic acid molecules useful in PCR-based methods, northern blotting and in situ hybridisation methods set out above in relation to the diagnostic methods of the invention.
  • Such agents may be able to selectively bind nucleic acid encoding FBXOIl polypeptide in which the residue corresponding to Q491 is mutated, for example to L; and/or in which the residue corresponding to S244 is mutated, for example to L
  • Agents that can be used in this aspect of the invention to determine the genotype of FBXOIl include primers, oligonucleotides, or other nucleic acid molecules useful in the methods set out above in relation to the diagnostic methods of the invention. Again, such agents may be able to selectively bind nucleic acid encoding FBXOIl polypeptide in which the residue corresponding to Q491 is mutated, for example to L; and/or in which the residue corresponding to S244 is mutated, for example to L
  • a further aspect of the invention provides an expression vector comprising a polynucleotide which encodes FBXOl 1 polypeptide.
  • a further aspect of the invention provides a host cell comprising the expression vector of the invention.
  • a - contains a poly-Adenylation signal S - sequence is a Suboptimal member of this cluster M - clone is putatively CDS-complete by MGC criteria
  • DNA encoding FBXOl 1 polypeptide may be expressed in a suitable host to produce a FBXOl 1 polypeptide.
  • the DNA encoding the FBXOl 1 polypeptide may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
  • Such techniques include those disclosed in US Patent Nos.
  • the DNA encoding the FBXOIl polypeptide may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the DNA insert may be operatively linked to an appropriate promoter.
  • Bacterial promoters include the E.coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage ⁇ PR and PL promoters, the phoA promoter and the trp promoter.
  • Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation (Hastings et al, International Patent No. WO 98/16643, published 23 April 1998).
  • the expression constructs may also desirably contain one or more sequence encoding a protein tag (for example a GST, Myc, or PolyHistidine tag) useful for identification and/or purification of the expressed polypeptide.
  • a protein tag for example a GST, Myc, or PolyHistidine tag
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that code for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanamycin or ampicillin resistance genes for culturing in E.coli and other bacteria.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA encoding FBXOl 1 polypeptide are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • the FBXOIl polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • bacteria eg. E.coli and Bacillus subtilis
  • yeasts eg. Saccaromyces cerevisiae
  • insect cell systems transformed with, for example, viral expression vectors (eg. baculovirus)
  • plant cell systems transfected with, for example viral or bacterial expression vectors
  • animal cell systems transfected with, for example, adenovirus expression vectors.
  • the vectors can include a prokaryotic replicon, such as the Col El ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E.coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA encoding FBXOI l polypeptide.
  • Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA); p7>c99A, pKK223-3, pKK233-3, ⁇ DR.540 and pRJT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells.
  • An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene. 25
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids ⁇ RS403, ⁇ RS404, ⁇ RS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPl, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls.
  • One such method involves ligation via homopolymer tails.
  • Homopolyrner polydA (or polydC) tails are added to exposed 3 ' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases.
  • the fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends j oined by DNA ligase.
  • Another method involves ligation via cohesive ends.
  • Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
  • a further method uses synthetic molecules called linkers and adaptors.
  • DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends.
  • Synthetic linkers pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini.
  • Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the FBXOl 1 polypeptide is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • a further aspect of the invention provides a method of making a FBXOIl polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative, the method comprising culturing a host cell as defined above which expresses said FBXOIl polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative, and isolating said polypeptide or a variant, fragment, derivative or fusion thereof or fusion of a said variant, or fragment or derivative.
  • the cell may be a eukaryotic cell, or may alternatively be a prokaryotic cell, though this may be less preferred.
  • a further aspect of the invention provides a gene therapy vector comprising a polynucleotide which encodes FBXOl 1 polypeptide.
  • Suitable vectors will be well known to those skilled in the art.
  • the vector may, for example, be a Moloney
  • LMV Leukaemia Virus
  • AAV adeno-associated vector
  • FIV-mediated gene therapy may be a route to treat chronic OM.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide encoding FBXOI l or FBXOI l polypeptide, or a gene therapy vector of the preceding aspect of the invention, and a pharmaceutically acceptable carrier.
  • a further aspect of the invention provides a polynucleotide encoding FBXOl 1 or FBXOIl polypeptide or a gene therapy vector of the invention or a pharmaceutical composition of the invention for use in medicine.
  • a further aspect of the invention provides the use of a polynucleotide encoding FBXOI l or FBXOI l polypeptide or a gene therapy vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the prevention or treatment of OM, for example the treatment or prevention of acute OM or chronic OM.
  • a further aspect of the invention provides the use of a modulator, for example an upregulator of expression, of an E3 ubiquitin ligase component in the manufacture of a medicament for the prevention or treatment of OM, for example the treatment or prevention of acute OM or chronic OM.
  • the modulator may be specific for an E3 ubiquitin ligase that comprises FBXOIl, but may alternatively have wider specificity, (for example modulating other SCF ubiquitin ligases, or modulating several or all E3 ubiquitin ligases) in which case it may be desirable for the medicament to be for delivery to the middle ear.
  • a further aspect of the invention provides a method of treating a patient with or at risk of developing OM comprising administering to the patient an appropriate quantity of a polynucleotide encoding FXBOl 1 or FBXOl 1 polypeptide or a gene therapy vector of the invention or a pharmaceutical composition of the invention.
  • a pharmaceutical composition of the invention, a polynucleotide encoding FXBOl 1 or FBXOl 1 polypeptide, or gene therapy vector of the invention may be used in combination with a means for delivery to the middle ear, as discussed above.
  • the composition or vector may be delivered using a tympanostomy tube (grommet).
  • the treatment may be administered alongside a further treatment for OM, for example a treatment as discussed above, for example a vaccine, antibiotic or protease inhibitor.
  • FBXOIl may act to down-regulate a pro-inflammatory cascade caused by the primary bacterial infection of the middle ear during acute OM.
  • This pro- inflammatory cascade may also be caused by viral infection, allergy, autoimmune disease or trauma to the middle ear.
  • Loss of function of FBXOl 1 may result in pathological levels of inflammation in the post acute OM middle ear.
  • inhibition of FBXOl l's interacting partners' / substrate(s) pro-inflammatory role or the up-regulation or replacement of FBXOl 1 is considered to be therapeutically useful.
  • a further aspect of the invention provides a method for generating a non-human animal which develops OM comprising reducing the amount of functional FBXOIl polypeptide, or reducing the amount of nucleic acid encoding said polypeptide.
  • the amount of functional FBXOl 1 polypeptide may be reduced by, for example, mutating one or more gene(s) encoding FBXOI l polypeptide by chemical or physical mutagenesis, or by homologous recombination or insertional mutagenesis, or using antisense or RNAi. Suitable techniques will be apparent to those skilled in the art.
  • the non-human animal may be a rodent, for example a mouse.
  • a further aspect of the invention provides a method for identifying a putative substrate polypeptide for FBXOl 1 polypeptide comprising the steps of:
  • the FBXOIl polypeptide may be provided in a form of a complex with other components of the SCF complex.
  • Methods for assessing protein-protein interactions will be well known to those skilled in the art, and include yeast two- hybrid (Y2H) and in vitro or in vivo pull-down techniques, for example co- immunoprecipitation techniques.
  • Y2H yeast two- hybrid
  • FBXOI l 's binding partners are not low molecular weight entities when running on conventional acrylamide gels
  • a further aspect of the invention provides a method for identifying a polypeptide involved in OM comprising:
  • step (ii) generating a non-human animal comprising a modified amount of the substrate polypeptide identified by step (ii), or modifying the amount of nucleic acid encoding said polypeptide;
  • step (iii) assessing whether the animal of step (ii) develops OM. If the modified animal develops OM (or a population of the modified animals shows a greater incidence of developing OM) then the polypeptide is considered to be involved in OM.
  • a further aspect of the invention provides a method of generating a non-human animal which develops OM (or is at greater risk than the unmodified animal of developing OM) comprising:
  • step (ii) generating a non-human animal comprising a modified amount of the substrate polypeptide identified by step (ii), or modifying the amount of nucleic acid encoding said polypeptide;
  • an interacting substrate polypeptide would be a standard experimental technique to those skilled in the art to create a knock out or knock in mutation of the said substrate using an engineered ES cell and thereby proceed to generate a rodent model that may develop OM.
  • a mutagen such as N-ethyl-N-nitrosourea (ENU)
  • ENU N-ethyl-N-nitrosourea
  • a further aspect of the invention provides an isolated or recombinant nucleic acid encoding FBXOl 1 (for example human or mouse FBXOl 1) in which the residue corresponding to Q491 is mutated, for example to L; and/or in which the residue corresponding to S244 is mutated, for example to L.
  • a further aspect of the invention provides an isolated or recombinant polypeptide having the amino acid sequence of FBXOI l (for example human or mouse FBXOIl) in which the residue corresponding to Q491 is mutated, for example to L; and/or in which the residue corresponding to S244 is mutated, for example to L.
  • a further aspect of the invention provides a kit of parts useful for assessing a patient's risk of developing otitis media (OM); or progression of OM; or for assisting in the diagnosis of OM, comprising one or more agents useful in determining one or more of: (a) the amount and/or function of FBXOIl polypeptide; (b) the amount of nucleic acid encoding FBXOI l; (c) the patient's genotype for FBXOIl; and, optionally, (d) a positive and/or negative control.
  • the kit may further comprise means for isolating protein and/or nucleic acid from a sample.
  • a further aspect of the invention provides a method for identifying a compound expected to be useful in the prevention or treatment of OM, for example the treatment or prevention of acute OM or chronic OM, the method comprising the steps of (i) assessing whether a test compound modulates the amount of nucleic acid encoding FBXOIl and/or the amount or function of FBXOIl polypeptide; and, optionally, (ii) selecting a compound that modulates the amount of nucleic acid encoding FBXOl 1 and/or the amount or function of FBXOl 1 polypeptide.
  • the selected compound may be a compound that increases the amount of nucleic acid encoding FBXOI l and/or the amount or function of FBXOI l polypeptide.
  • the selected compound may alternatively be a compound that decreases the amount of nucleic acid encoding FBXOIl and/or the amount or function of FBXOIl polypeptide. .
  • the method may be performed in vitro, for example using a cell extract or purified and/or recombinant components, example when assessing whether the test compound modulates the activity of FBXOI l. Methods for assessing FBXOl 1 activity are discussed above.
  • the method may be performed as a cell-based assay, either when assessing FBXOl 1 activity or when assessing effects on expression of FBXOl 1.
  • the cells may be in an in vitro cell culture or in a test non-human animal.
  • middle ear epithelial cells lines such as those described by Herman et al. J Cell Physiol. 1993 Mar; 154(3):615-22, or Jin et al. Ann Otol Rhinol Laryngol. 1999 Oct;108(10):934-43 and could be engineered using techniques familiar to those skilled in the art such that they contained a construct that reported the expression of FBXOI l.
  • the FBXOIl protein could be tagged, for example, with a fluorescent marker. The marker could be used to report the effects of small molecules, for example, and whether they cause increased or decreased expression of FBXOIl.
  • reporter protein or reporter RNA
  • reporter RNA expression of a "reporter” protein (or reporter RNA), as well known to those skilled in the art may alternatively or in addition be assessed (for example a recombinant construct may be used in which regulatory regions of the FBXOl 1 gene regulate expression of a non-FBXOl 1 coding region).
  • the reporter protein may be one whose activity may easily be assayed, for example ⁇ -galactosidase, chloramphenicol acetyltransferase or luciferase.
  • the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions.
  • WST-I is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
  • the unengineered aforementioned cell lines could also be used in tandem with in vitro activity assays, for example ubiquitinylation assays, to determine whether screened compounds affect the FBXOl 1 -containing E3 ligases.
  • test compound may be a small molecule, polypeptide or genetic construct, as will be well known to those skilled in the art, and as discussed above.
  • Compounds identified in the methods may themselves be useful as a drag or they may represent lead compounds for the design and synthesis of more efficacious compounds,
  • the compound may be a drug-like compound or lead compound for the development of a drag-like compound for each of the above methods of identifying a compound. It will be appreciated that the said methods may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non- selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • screening assays which are capable of high throughput operation, for example in microtitre plates, are particularly preferred.
  • Enzyme assays may be used, as discussed above. Further examples may include cell based assays and protein-protein binding assays. It will be understood that it could be desirable to identify compounds that may modulate the activity of FBXOI l or an FBXOI l -containing E3 ligase in vivo.
  • reagents and conditions used in the method may be chosen such that the interactions between, for example, the E3 ligase and the substrate, are similar to those between the human E3 ligase and a naturally occuring substrate.
  • assay systems may be used to assess a compound, in some of which the convenience of the assay or the specificity for an effect on the FBXOI l or FBXO 11 -containing E3 ligase may be optimised, whilst in others the in vivo relevance may be optimised, for example by assessing the effect of the compound in a whole cell.
  • the compounds that are tested in the screening methods of the assay or in other assays in which the ability of a compound to modulate the FBXOl 1 activity may be measured may be compounds that have been selected and/or designed (including modified) using molecular modelling techniques, for example using computer techniques, or may be compounds selected on the basis of reported activity against other ubiquitin-protein ligase(s).
  • the screening method may further comprise the step of assessing the effect of the compound in an animal model of OM, for example in the Jeff mouse or in an animal model in which the amount of functional FBXOIl polypeptide is reduced, for example by mutation of one or more genes encoding FBXOl 1.
  • the screening method may further comprise the step of synthesising, purifying and/or formulating the selected compound.
  • the compound may be formulated for pharmaceutical use, for example for use in in vivo trials in animals or humans.
  • the invention provides a method for preparing a compound which may be useful in the treatment or prevention of OM, for example acute OM or chronic OM, the method comprising 1) performing a screening method of the invention and 2) synthesising, purifying and/or formulating the selected compound.
  • the molecule consists of an F-box motif, 2 carbohydrate binding (CASH) domains and a zinc finger domain (ZnF). The amino acid numbers constituting these domains are shown below the appropriate domain.
  • the position of the Jeff and Mutt mutations are shown (arrows). Peptides flanking the Jeff mutation were used to raise 2 antibodies to the central part of the molecule.
  • C-rYVHEKGRGQFIEN Pl; SEQ ID No: 1
  • CPIVRHNKIHDGQHG P2; SEQ ID No: 2
  • Polyclonal antibodies were raised by covalAbUK, www.covalab.com. These antibodies are rabbit-antimouse.
  • FIG. 1 Coronal sections of the head (H&E staining) demonstrating the palate and facial phenotypes of late embryonic Jeff homozygotes, and perinatal Mutt homozygotes and Jeff/Mutt compound heterozygotes that fail to thrive. Scale bar,
  • Facial clefts are marked with a star and cleft palate are marked with an arrow.
  • Antibody staining of various tissues was carried out using an FBXOI l antibody with a standard DAB staining protocol according to manufacturer's instructions and micro waving in water as an antigen retrieval technique (see text and Figure 1).
  • Antibody labelling of Fbxoll was completed ablated by peptide competition on both Western blots of protein from newborn head tissue and on tissue sections, indicating antibody specificity (data not shown).
  • Figure 4 Alignment of human and mouse FBXOIl sequences Alignment of human (SEQ ID No: 25) and mouse (SEQ ID No: 26) FBXOl 1 polypeptide sequences.
  • the top sequence is human (Ensemble sequence ID ENSP00000323822), and the bottom sequence is mouse (Ensemble sequence ID ENSMUP00000005504. The location of the Jeff and Mutt mutations are shown. The sequences shown are wild-type.
  • Figure 5 Mouse Jeff mutations polypeptide sequence (A; SEQ ID No: 27) and polynucleotide sequence (B; SEQ ID No: 28). ⁇
  • mouse Fbxoll polypeptide sequence shown in Figure 5 A begins with the Methionine which corresponds to the ATG codon at the beginning of the transcript. However, there are an additional five residues at the beginning of the mouse FBXOIl polypeptide sequence used in Figure 4 (SEQ ID No 26) which is from Ensemble sequence ID ENSMUP00000005504.
  • Figure 6 Human FBXOIl wild-type polypeptide sequence (A; SEQ ID No: 25) and polynucleotide sequence (B; SEQ ID No: 29).
  • Example 1 A mutation in the F-box gene, Fbxoll causes Otitis Media in the Jeff mouse.
  • Otitis media inflammation of the middle ear mucosa, is the most common cause of hearing impairment and surgery in children.
  • Recurrent (ROM) and chronic (COME) forms of otitis media are known to have a strong genetic component, but nothing is known of the underlying genes involved in the human population.
  • the inventors have previously identified a novel semi-dominant mouse mutant, Jeff, in which the heterozygotes develop chronic suppurative otitis media [1] and represent a model for chronic forms of otitis media in humans.
  • the inventors now show that Jeff carries a mutation in the F-box gene Fbxoll, which is expressed in the mucin secreting epithelial cells of the middle ear from late embryonic stages through to day 13 of postnatal life.
  • Otitis media (OM) 5 inflammation of the middle ear is the most common cause of hearing impairment in children [2,3]. In addition, it remains the commonest cause for surgery in children in the developed world. Acute episodes of OM are usually associated with middle ear infections by the bacterial pathogens Streptococcus pneumoniae and Haemophilus influenzae [4]. However, prolonged stimulation of the inflammatory response accompanied by poor mucociliary response can lead to a persistent middle ear effusion (OME) and in many children, recurrent or chronic suppurative forms of the disease may develop. The prevalence of OM along with its recurrent and chronic nature underlies the frequency of tympanostomies undertaken in affected children.
  • OME middle ear effusion
  • the inventors have proceeded to identify the gene underlying the Je/f mutant.
  • Jeff carries a mutation in the F-box gene, Fbxoll.
  • the inventors have isolated and characterized an additional ENU mutant allele at this locus, the Mutt mutation.
  • Both Jeff and Mutt homozygotes demonstrate cleft palate defects, facial clefting and perinatal lethality.
  • Fbxoll represents an important candidate gene for the study of the genetic pathways involved in OM in the human population.
  • the role of Fbxoll in the development of the palatal shelves implicates this as an important candidate for studies of cleft palate in the human population.
  • mice were housed in conventional cages and were provided with food and water ad libitum and maintained according to Home Office and ethical regulations. Sentinel health screening from this old MRC Harwell mouse house showed presence of the following FELASA listed agents [http://www.felasa.org] (31): MHV (judged by histology to be enteropathic strains), Adenovirus II and TMEV none of which are primary respiratory pathogens. Intestinal flagellates, pinwo ⁇ ns and the opportunist respiratory pathogen Pasteurella pneumotropica were also common isolates.
  • the Jeff colony has subsequently been rederived by embryo transfer into a new MRC Harwell SPF facility, the Mary Lyon Centre, and is maintained on C57BL/6J background.
  • the stocks are free of FELASA listed agents, but the nasopharynx of sentinel mice have similar non-FELASA list streptococcal and staphylococcal flora.
  • ENU mutagenesis was carried out on a BALB/c background and males outcrossed to C3H/HeN (10).
  • affected Fl individuals were backcrossed to C3H/HeN.
  • the inventors originally mapped the Jeff mutation to chromosome 17, based on 30 affected individuals (1).
  • 920 meioses were generated. Mapping was further enhanced by a second backcross ([Jf/+ x C57BL/6J] x C57BL/6J]). Markers and primer sequences are available on request from the inventors.
  • DPLC Denaturing high-performance liquid chromatography
  • Mutation detection was performed using a Transgenomic wave machine, utilizing
  • DHPLC DHPLC.
  • the system was run according to manufacturer's instructions (Transgenomic). Exons to be screened were amplified using primers placed in flanking introns. DNAs from a Jf/+ mouse and a BALB/c (+/+) mouse were amplified for each exon using Taq polymerase (ABgene), at an annealing temperature of 55 0 C. Following amplification, heteroduplexes were formed by thermocycling.
  • ABgene Taq polymerase
  • PCR products from Jf/+ and BALB/c DNA were purified using QIAquick PCR purification kit (Qiagen). Direct sequencing was performed using Applied Biosystem's Bigdye Terminator v3.1 cycle sequencing mix and sequenced on an ABI prism 377 DNA sequencer according to manufacturer's instructions.
  • Genotyping for Jeff mice was performed by PCR amplification of the exon containing the mutation followed by digest with BcII. Amplification of Exon 13 using primers: 5 ' TGC CTG ATG TAA AAA TTA CTC CAC 3 ' (SEQ ID No: 30) and 5' TCT CTA GGG ATC AGG CAC ATC 3' (SEQ ID No: 31), yields a product of 199bp. In the presence of the mutation, a BcII restriction site is introduced giving 2 bands of 132bp and 67bp.
  • PCR product (77bp) and primer: 5' TCA TCA TTG AGA ACA CTA GA S' (SEQ ID No: 34) were used for subsequent pyrosequencing SNP analysis to identify differences in sequences.
  • Fbxoll polyclonal antibody A polyclonal antibody against mouse FBXOI l was produced by CovalabUK (www.covalab.com ' ) using two peptides as antigens.
  • the peptide sequences were CIYVHEKGRGQFIEN (residues 419-433; SEQ ID No: 1) and CP ⁇ VRHNKIHDGQHG (residues 497-511; SEQ ID No: 2) and they lay in the central unique part of the mouse Fbxol l protein. Both peptides were injected together into rabbits.
  • the serum from the immunised animals was collected and the antibody was purified by affinity chromatography using the peptides.
  • Affinity purified antibody was tested on Western Blot using whole mouse head lysates where it recognises bands of approximately 46KDa, 32KDa and 26KDa. Various cell lines showed bands of similar sizes by Western Blot. Since the antibody does not detect full-length protein we tested its specificity in various ways. Firstly, preincubation of the affinity purified antibody with increasing amounts of the peptides used as antigens for its production gradually abolished the signal detected by Western Blot and immunohistochemistry. Secondly, following immunoprecipitation with the purified antibody, the 46KDa band was digested with trypsin and identified as FBXOIl by peptide mass fingerprint (data not shown).
  • the inventors transfected Cos-7 cells with a plasmid containing the mouse FBXOIl sequence tagged to the Xpress epitope (Invitrogen).
  • a plasmid containing the mouse FBXOIl sequence tagged to the Xpress epitope Invitrogen.
  • anti-Fbxoll polyclonal antibody recognises an identical band to that detected by anti-Xpress antibody and of the expected size (approximately 94KDa).
  • Mouse head tissue was homogenized at 4 0 C in lysis buffer (5OmM HEPES, PH7.4, 10% Triton X-IOO, 5OmM Sodium Phosphate, 1OmM EDTA, 1OmM sodium fluoride, 1OmM Sodium Orthovanadate, 2mM Benzamidine and a protease inhibitor cocktail). Homogenate was solubilised on ice for 1 hour and centrifuged at 4 0 C, first at 3000rpm for 15 minutes and then at 14000 for 1 hour at 4 0 C. The supernatant was run in a 4-12% acrylamide gel (Invitrogen NuPAGE) and then the gel was blotted onto a PVDF membrane.
  • lysis buffer 5OmM HEPES, PH7.4, 10% Triton X-IOO, 5OmM Sodium Phosphate, 1OmM EDTA, 1OmM sodium fluoride, 1OmM Sodium Orthovanadate,
  • the membrane was incubated in a blocking solution containing PBS, 0.1% Tween 20 and 5% skimmed milk for one hour at room temperature. After blocking, the membrane was incubated with anti-Fbxol 1 antibody at 7.5 ⁇ g/ml in blocking solution for one hour at room temperature. After four washes in PBS/0.1% Tween 20 the membrane was incubated with anti-rabbit IgG conjugated to HRP for one hour at room temperature and washed as before. The bands labelled by the antibody were detected by ECL-Plus (GE Healthcare). Histology.
  • the ears and palates were surveyed in SPF Jeff heterozygotes on C57BL/6J background (two 5DAB, two 13DAB, three 28DAB, five 56DAB, five 120DAB). These heads were decalcified 24-48h with Immunocal (Decal Corp. Tallman NY) and stained with H&E.
  • the avidin-biotin complex (ABC) method was used for immunohistochemical analysis. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in isopropanol for 20min.
  • the slides were pre-treated by boiling in a microwave in ImM EDTA pH 8 for E8.5-13.5 embryos and in water, for E14.5-18.5 embryos and all adult tissues, for 14min, cooled at RT for 20min and rinsed with phosphate-buffered saline.
  • the immunostaining was performed using a DAKO autostainer at room temperature. To inhibit non-specific endogenous biotin staining the DAKO Biotin Blocking System was used (DAKO, X0590).
  • a blocking solution of 10% swine serum (DAKO, X 0901) was used for one hour. Fbxoll antibody incubations were conducted for one hour using a 1:200 dilution. Biotinylated swine anti-rabbit antibody (DAKO, E 0353) and ChemMate Detection Kit (DAKO, K 5001) were used to develop the specific signals. Negative control sections were stained with the Fbxoll antibody previously incubated with the blocking peptides and processed identically. The slides were counterstained with haematoxylin. Results
  • the Jeff mutant is a semi-dominant mutation with the heterozygote previously described as having chronic proliferative otitis media [I]. Mice homozygous for the Jeff mutation demonstrated perinatal lethality, dying at birth or within a few hours of birth. Homozygotes are born with upper eyelids open and show clefting of the hard or soft palate.
  • the Jeff mutation was mapped using backcrosses to an approximately 300kb region of distal chromosome 17 flanked by the markers SNPMGUCl 7 and Dl 7MtI (see Methods; data not shown). Based on Ensembl predictions (http://www.ensembl.org ' ) this region contains 3 genes and 2 pseudogenes. The 3 genes in the region are Fbxoll, Msh ⁇ and a novel transcript, the 4OS ribosomal s24 gene.
  • the change occurs in a highly conserved region of this protein that has been maintained through evolution ( Figure 1C).
  • the human Fbxoll locus has one predicted transcript and encodes a protein of 843 amino-acids, coded for in 22 exons.
  • the mouse protein is 850 residues in length.
  • the 94KDa protein consists of 2 carbohydrate binding domains as well as an F-box motif and a zinc-finger domain (Figure IA).
  • Homozygotes are born with upper eyelids open and show clefting of the hard or soft palate as well as facial clefting ( Figure 2B).
  • the inventors used DNA and sperm archives derived from ENU mutagenesis programmes [11] to identify an additional allele at the Fbxoll locus.
  • the inventors screened the first 7 exons of Fbxoll employing heteroduplex analysis of 4200 mutant mice and identified a further point mutation leading to a serine to leucine change, S244L (Figure IA) in a conserved region of the protein ( Figure IB).
  • This second allele of Fbxoll, Mutt was rederived and the heterozygous and homozygous phenotypes were examined.
  • the developing secondary palate is now also expressing in the nasal, medial and oral epithelia of the palatal shelves as they elevate above the tongue (Figure 3C).
  • Figure 3C expression can be seen in the lung, kidney, heart, liver, muscle and adrenal gland.
  • fusion of the shelves has occurred, with expression in the nasal ( Figure 31) and oral epithelia.
  • Figure 3D expression in the lung is confined to the bronchial epithelial cells ( Figure 3D) and expression is evident in the bone marrow, skin, tissue macrophages, osteoblasts, kidney liver and spleen.
  • FIG. 8.5 bone marrow, liver, kidney and muscle are positive, but expression in heart and lung is beginning to fade out.
  • the Jeff mouse mutant is a model of chronic OM in the human population.
  • the inventors have therefore proceeded to map and identify the mutation underlying the Jeff mutant.
  • the Jeff mutant carries a mutation in the F-box protein, Fbxoll.
  • FBXOIl is expressed in the middle ear epithelium just preceding the period OM is evident in the Ji ⁇ /f mouse.
  • the inventors have also isolated and characterized an additional mutant allele at the Fbxoll locus, the Mutt mutation.
  • Fbxoll is one member of a large family of proteins involved with ubiquitination. Much of the targeted protein ubiquitination that occurs in eukaryotic organisms is performed by cullin-based E3 ubiquitin ligases, which form a superfamily of modular E3s [12].
  • the best understood cullin-based E3 is the SCF ubiquitin ligase [13-15] composed of a modular E3 core, containing CULl and RBXl, SKPl and a member of the F-box family of proteins [16-21]. The interaction of the F-box protein with SKPl occurs via the F-box motif, an approximately 40 amino-acid motif first identified in yeast and human cyclin-F.
  • F-box proteins also contain further interaction domains that bind ubiquitination targets.
  • a recent study [12] identified 74 mouse genes encoding recognizable F-box motifs subdivided into 3 subsets: FBXL (containing leucine rich repeats); FBXW (containing WD40 motifs) and FBXO (proteins that contain an F-box and an 'other' identifiable motif) of which there are 47 members to date [12.22].
  • a fragment of FBXOIl was originally identified in a differential expression analysis of cultured melanocytes from generalized vitiligo patients versus control cells [23]. This cDNA which they called VITl was found to be absent in melanocytes from vitiligo patients.
  • Recently FBXOl 1/PRMT9 was identified as a arginine 40
  • the Mutt allele does not develop OM, suggesting it is a hypomorphic allele at tfie Fbxoll locus. However, like the a proportion of Mutt homozygotes show facial clefting and cleft palate. At E 14.5 FBXOIl is expressed in the margins of the fusing palatal shelves.
  • the mutational and expression analysis of the Fbxoll gene identifies a new locus involved with cleft palate and facial clefts in the mouse [27-30], and which is also likely to be relevant to cleft palate defects in the human population.
  • Cdc53 is a scaffold protein for multiple Cdc34/Sk ⁇ l/F-box protein complexes that regulate cell division and methionine biosynthesis in yeast. Genes Dev., 12, 692-705.
  • Cdc53/cullin and the essential Hrtl RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev., 13, 1614-1626.
  • ROCl a homolog of APCIl, represents a family of cullin partners with an associated ubiquitin ligase activity. MoI. Cell, 3, 535-541.

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Abstract

L'invention concerne un procédé pour évaluer le risque d'un patient de développer une otite moyenne (MO) ou la progression d'une otite moyenne ; pour offrir une aide au diagnostic de l'otite moyenne, ledit procédé comprenant les étapes suivantes : (i) obtention d'un échantillon contenant un acide nucléique et/ou une protéine provenant d'un patient ; et (ii) détermination de : a) la quantité et/ou la fonction du polypeptide FBXO11 ; b) de la quantité d'acide nucléique codant le FBXO11 ; c) du génotype du patient pour le FBXO11.
PCT/GB2006/003731 2005-10-06 2006-10-06 Association du gene fbx011 a l'otite moyenne Ceased WO2007039759A1 (fr)

Applications Claiming Priority (2)

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GB0520311.2 2005-10-06
GB0520311A GB0520311D0 (en) 2005-10-06 2005-10-06 Methods

Publications (1)

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WO2007039759A1 true WO2007039759A1 (fr) 2007-04-12

Family

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PCT/GB2006/003731 Ceased WO2007039759A1 (fr) 2005-10-06 2006-10-06 Association du gene fbx011 a l'otite moyenne

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GB (1) GB0520311D0 (fr)
WO (1) WO2007039759A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055417A1 (fr) * 2000-01-26 2001-08-02 Biodoor Gene Technology Ltd. Shanghai Nouveau polypeptide, proteine a f-box 65, et polynucleotide codant pour ce polypeptide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055417A1 (fr) * 2000-01-26 2001-08-02 Biodoor Gene Technology Ltd. Shanghai Nouveau polypeptide, proteine a f-box 65, et polynucleotide codant pour ce polypeptide

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF OTOLARYNGOLOGY - HEAD AND NECK SURGERY 2006 UNITED STATES, vol. 132, no. 7, 2006, pages 729 - 733 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; March 2002 (2002-03-01), LIU HAI-YAN ET AL: "Screening and identification of human lung cancer-related antigens", XP002415262, Database accession no. PREV200200258512 *
DATABASE EMBASE [online] ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL; 2006, SEGADE F ET AL: "Association of the FBXO11 gene with chronic otitis media with effusion and recurrent otitis media: The Minnesota COME/ROM family study", XP002415263, Database accession no. EMB-2006346811 *
DATABASE WPI Week 200151, Derwent World Patents Index; AN 2001-476219, XP002415345 *
HARDISTY RACHEL E ET AL: "The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media.", JARO JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY, vol. 4, no. 2, 2003, pages 130 - 138, XP002415259 *
HARDISTY-HUGHES R E ET AL: "A mutation in the F-box gene, Fbxo11, causes otitis media in the Jeff mouse", HUMAN MOLECULAR GENETICS, vol. 15, no. 22, 11 October 2006 (2006-10-11), pages 3273 - 3279, XP002415261 *
LE POOLE I C ET AL: "'VIT1', a novel gene associated with vitiligo", PIGMENT CELL RESEARCH, vol. 14, no. 6, 2001, pages 475 - 484, XP002415260 *
SHENGWU HUAXUE YU SHENGWU WULI XUEBAO, vol. 34, no. 2, 2002, pages 171 - 175 *
ZHENG ET AL: "Mouse models as a tool to unravel the genetic basis for human otitis media", BRAIN RESEARCH, vol. 1091, no. 1, 26 May 2006 (2006-05-26), pages 9 - 15, XP005535259 *

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