EP1863927A2 - Compositions et methodes destinees a traiter et a diagnostiquer des troubles inflammatoires - Google Patents

Compositions et methodes destinees a traiter et a diagnostiquer des troubles inflammatoires

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Publication number
EP1863927A2
EP1863927A2 EP06725045A EP06725045A EP1863927A2 EP 1863927 A2 EP1863927 A2 EP 1863927A2 EP 06725045 A EP06725045 A EP 06725045A EP 06725045 A EP06725045 A EP 06725045A EP 1863927 A2 EP1863927 A2 EP 1863927A2
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EP
European Patent Office
Prior art keywords
seq
nrgl
polypeptide
psoriasis
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP06725045A
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German (de)
English (en)
Inventor
Ilya Chumakov
Daniel Cohen
Fabio Macciardi
Benedicte Belloir
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Ares Trading SA
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Ares Trading SA
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Priority to EP06725045A priority Critical patent/EP1863927A2/fr
Publication of EP1863927A2 publication Critical patent/EP1863927A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates, generally, to methods and compositions for detecting or treating inflammatory disorders.
  • the inflammatory disorder may be an inflammatory CNS disorder such as multiple sclerosis (MS), an inflammatory disorder involving multiple organ systems as in the case of systemic lupus erythematosus (SLE) or an inflammatory skin disorder such as psoriasis.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • psoriasis inflammatory skin disorder
  • the present invention more particularly relates to the human NRGl gene, which can be used for the diagnosis, prevention and treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders, as well as for the screening of therapeutically active drugs.
  • the invention further discloses specific polymorphisms, splice variants or alleles of the NRGl gene that are related to inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus or psoriasis, as well as diagnostic tools and kits based on these susceptibility alterations.
  • the invention can be used in the diagnosis or detection of the presence, risk or predisposition to, as well as in the prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders.
  • Psoriasis is considered mild if it affects less than 5% of the surface of the body, moderate if 5-30% of the skin is involved, and severe if the disease affects more than 30% of the body surface.
  • the invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in any mammalian subjects, particularly human patients.
  • the invention relates to antibodies, which specifically bind to the NRGl polypeptide.
  • multi sclerosis may be defined as in the DSM-IV classification (Diagnosis and Statistical Manual of Inflammatory CNS Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994).
  • systemic lupus erythematosus may be defined as by Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF et al. "The 1982 revised criteria for the classification of Systemic lupus erythematosus” Arthritis Rheum 1982;25: 1271 -1277.
  • Duchenne muscular dystrophy is a fatal disorder caused by absence of dystrophin.
  • Krag et al. PNAS, September 2004; 101(38):13856-60
  • NRGl isoform 6 tested the ability of NRGl isoform 6 to improve the dystrophic phenotype in the mdx mouse model of DMD.
  • Intraperitoneal injections of the small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in upregulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction-mediated damage, and a reduction of muscle pathology.
  • gene as used herein shall be construed to include any type of coding nucleic acid region, including genomic DNA (gDNA), complementary DNA (cDNA), synthetic or semi- synthetic DNA, any form of corresponding RNA (e.g., mRNA), etc., as well as non coding sequences, such as introns, 5'- or 3 '-untranslated sequences or regulatory sequences (e.g., promoter or enhancer), etc.
  • the term gene particularly includes recombinant nucleic acids, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences.
  • a gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. Genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. The term “gene” may comprise any and all splicing variants of said gene.
  • the present invention also encompasses fragments of the NRGl gene.
  • a fragment of a gene designates any portion of at least about 8 consecutive nucleotides of a sequence of said gene, preferably at least about 15, more preferably at least about 25 nucleotides, further preferably of at least 35, 50, 75, 100, 150, 200 or 300 nucleotides. Fragments include more particularly all possible nucleotide length between 8 and 500 nucleotides, preferably between 15 and 300, more preferably between 25 and 200.
  • the present invention also encompasses DNA sequences, which have at least 40% identity with the NRGl sequence. More preferably, they have at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity thereto.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • the present invention also encompasses DNA sequences, which hybridize to the complement of the NRGl nucleic acid sequence under moderately stringent conditions or under highly stringent conditions.
  • stringent conditions refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et ah, Current Protocols in Molecular Biology, supra, Interscience, N.Y., ⁇ 6.3 and 6.4 (1987, 1992), and Sambrook et a (Sambrook, J. C, Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes.
  • stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
  • a polypeptide designates any protein or polypeptide encoded by the NRGl gene as disclosed above, respectively.
  • polypeptide designates, within the context of this invention, a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • a fragment of a polypeptide designates any portion of at least 8 consecutive amino acids of a sequence of said protein, preferably of at least about 15, more preferably of at least about 20, further preferably of at least 50, 100, 250, 300 or 350 amino acids.
  • polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides variants which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • the present invention encompasses all polypeptide sequences defined in Figures 2 to 11.
  • the NRGl polypeptide according to this invention has the sequence defined in SEQ ID NO. 1.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 2.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 3.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO. 7.
  • the NRGl polypeptide according to this invention has the sequence as defined in SEQ ID NO.10.
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BPl 6 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located between the NRGl polypeptide-encoding sequence and the heterologous protein sequence, so that the NRGl polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by Standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences for NRGl in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • treat or “treating” as used herein is meant to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.
  • treatment as used herein also encompasses the term “prevention of the disorder”, which is, e.g., manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. Treatment of the disorder is, e.g., manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
  • modulated or modulation or regulated or “regulation” as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating)] and downregulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
  • oligonucleotides and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide is used herein as an adjective to describe compounds comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a compound, or individual unit in a larger nucleic acid compound, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO95/04064, the disclosure of which is incorporated herein by reference.
  • the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides.
  • the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • primer denotes a specific oligonucleotide sequence, which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
  • a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
  • Typical primers of this invention are single-stranded nucleic acid molecules of about 6 to 50 nucleotides in length, more preferably of about 8 to about 40 nucleotides in length, typically of about 16 to 25.
  • the Tm is typically of about 60°C or more.
  • the sequence of the primer can be derived directly from the sequence of the target gene. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
  • Probes of this invention typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 750, more preferably of between 15 and 600, typically of between 20 and 400.
  • the sequence of the probes can be derived from the sequences of the NRGl gene sequence.
  • the probe may contain nucleotide substitutions and/or chemical modifications, e.g., to increase the stability of hybrids or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, etc.
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • non-human animal refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • twin and phenotype are used interchangeably herein and refer to any clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example.
  • the terms “trait” or “phenotype” are used herein to refer to symptoms of, or susceptibility to inflammatory disorder; or to refer to an individual's response to an agent acting on inflammatory disorder; or to refer to symptoms of, or susceptibility to side effects to an agent acting on inflammatory disorder.
  • allele refers to one of the variant forms of a biallelic or multiallelic alteration, differing from other forms in its nucleotide sequence. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a "polymorphic site” is the locus at which the variation occurs.
  • a polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides.
  • a single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site.
  • SNP single nucleotide polymorphism
  • antibody encompasses monoclonal and polyclonal antibodies, chimeric, humanized, fully human, bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies, as further explained below.
  • the term "selective" binding indicates that the antibodies preferentially bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope. In other words, binding to the target polypeptide can be discriminated from non-specific binding to other antigens.
  • Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity.
  • the term fragment includes any binding portion of an antibody, such as Fab, F(ab')2, CDR domains, etc.
  • Derivatives include human or humanized antibodies, polyfunctional antibodies, single-chain antibodies (e.g., ScFv), etc. Methods for producing antibodies, fragments or derivatives thereof are well known in the art, including immunization of an animal and collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with appropriate cell lines).
  • Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495), incorporated therein by reference. Briefly, these methods comprise immunizing an animal with the antigen, subsequently recovering spleen cells and fusing these cells with immortalized cells, such as myeloma cells, to produce hybridomas. Hybrodimas producing the desired monoclonal antibodies can be selected by limit dilutions. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544).
  • the antibodies may be coupled to heterologous moieties, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers.
  • heterologous moieties such as toxins, labels, drugs or other therapeutic agents
  • the present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis, systemic lupus erythematosus, psoriasis and related disorders in a human subject.
  • the present methods may be implemented at various development stages of said pathologies, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth.
  • the invention is suited to determine the prognosis, to assess a predisposition to or a risk of development of pathology, to characterize the status of a disease or to define the most appropriate treatment regimen for a patient.
  • a particular object of this invention resides in a method of detecting the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject, the method comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in said subject.
  • Another object of this invention relates to methods of assessing the response of a subject to a treatment of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, the methods comprising detecting the presence of an alteration in a NRGl gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
  • interferon and "interferon-beta (IFN-beta)", as used herein, are intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
  • IFN-beta suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering).
  • Rebif® Sterotono
  • Avonex® Biogen
  • Betaferon® Schering
  • the use of interferons of human origin is also preferred in accordance with the present invention.
  • the term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.
  • Rebif® (recombinant human interferon-) is the latest development in interferon therapy for multiple sclerosis (MS) and represents a significant advance in treatment.
  • Rebif® is interferon (IFN)-beta Ia, produced from mammalian cell lines. It was established that interferon beta- Ia given subcutaneously three times per week is efficacious in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS).
  • Interferon beta- Ia can have a positive effect on the long-term course of MS by reducing number and severity of relapses and reducing the burden of the disease and disease activity as measured by MRI.
  • IFN is recombinant IFN- ⁇ Ib produced in E. CoIi, commercially available under the trademark Betaseron
  • IFN may preferably be administered sub-cutaneously every second day at a dosage of about of 250 to 300 g or 8 MIU to 9.6 MIU per person.
  • IFN is recombinant IFN- ⁇ Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Avonex
  • IFN may preferably be administered intra-muscularly once a week at a dosage of about of 3Og to 33 g or 6 MIU to 6.6 MIU per person.
  • Quinacrine in a dosage of 100 mg per day, can be added without increasing the risk of retinopathy, or the patient can be switched to chloroquine HCl (Aralen), in a dosage of 250 mg per day.
  • chloroquine HCl Alen
  • lupus nephritis are severe. Patients with milder forms, including mesangial glomerulonephritis and focal proliferative glomerulonephritis, may respond to corticosteroid therapy alone or with steroid-sparing drugs such as azathioprine. Cyclophosphamide (Cytoxan) is more effective than corticosteroids alone for the treatment of severe forms of lupus nephritis (diffuse proliferative glomerulonephritis). One of the major complications of systemic lupus erythematosus is premature or accelerated atherosclerosis.
  • Corticosteroid treatment increases the levels of cardiovascular risk factors, including weight, blood pressure, cholesterol and homocysteine levels.
  • Patients with systemic lupus erythematosus who have already had a manifestation of antiphospholipid antibody syndrome require treatment. Patients who have had venous or arterial thrombosis appear to benefit from maintenance therapy with high- intensity (International Normalized Ratio of 3 to 4) warfarin (Coumadin).
  • the most frequent hematologic manifestation of systemic lupus erythematosus is anemia, usually normochromic normocystic, reflecting chronic illness.
  • Anemia in patients with systemic lupus erythematosus is most often associated with chronic disease or is related to iron deficiency.
  • Classic autoimmune hemolytic anemia can present acutely (and severely) or as a chronic condition. Severe hemolytic anemia is treated initially with intravenous methylprednisolone, 1,000 mg per day for three days.
  • Treatment approaches for psoriasis depend on the severity of the symptoms. In mild forms of psoriasis, topical treatments are applied, such as vitamin D3 analogs, corticosteroids, retinoids and coal tar. Oily bath solutions and moisturizers have a soothing effect on the skin.
  • Moderate to severe psoriasis is usually treated with light therapy. This includes therapy by sunlight or Ultraviolet B. Also available are combination therapies combining oral or topical administration of Psoralen with exposure to Ultraviolet A. Light therapy may also be combined with administration of retinoids.
  • efalizumab (marketed under the tradename Raptiva) is a humanised monoclonal antibody, which is recombinantly produced in mammalian cells. It inhibits activation of T cells and reduces inflammation in psoriasis.
  • alteration in a NRGl gene or polypeptide may be any nucleotide or amino acid alteration associated to multiple sclerosis, systemic lupus erythematosus or a related disease.
  • a susceptibility alteration in the NRGl gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertion(s) in the coding and/or non-coding region of the gene, either isolated or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene. Typical deletions affect small regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene.
  • Rearrangements include for instance sequence inversions.
  • An alteration in the NRGl gene may also be an aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA, allelic loss of the gene or allelic gain of the gene.
  • the alteration may be silent (i.e., create no modification in the amino acid sequence of the protein), or may result, for instance, in amino acid substitutions, frameshift mutations, stop codons, RNA splicing, e.g. the presence of a non-wild type splicing pattern of a messenger RNA transcript, or RNA or protein instability or a non-wild type level of the NRGl polypeptide.
  • the alteration may result in the production of a polypeptide with altered function or stability, or cause a reduction or increase in protein expression levels.
  • Typical susceptibility alterations are single nucleotide polymorphisms (SNPs).
  • NRGl gene which are associated with multiple sclerosis, systemic lupus erythematosus or psoriasis. These mutations are reported in table 2 (tables 2a, 2b, 2c, 2d, 2e). In summary, the association results of the single biallelic alteration frequency analysis show that the NRGl gene is associated with multiple sclerosis, systemic lupus erythematosus and psoriasis.
  • the most preferred alterations linked to SLE or psoriasis are those SNP for which the Mantel-Haenszel statistical test was significant. This means that the allele frequency difference between cases and controls is significant for these SNPs when the two SLE or psoriasis sets of data are analysed simultaneously and stratified by study populations. For MS, the Mantel-Haenszel statistical test was not significant for any of the SNPs; this means, that different SNPs are linked to MS in the different populations.
  • Base 1 base number 119199117 of chromosome 3 to base number 117011800
  • a preferred embodiment of the present invention comprises the detection of the presence of a susceptibility alteration as disclosed in Tables 2a, 2b or 2c in the NRGl gene or RNA sequence of a subject, more particularly the detection of at least one alteration as disclosed in Table 2d or 2e, or any combination thereof.
  • Another preferred object of this invention is a method of detecting the presence of or predisposition to psoriasis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2c or 2e in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to psoriasis or a related disorder in said subject.
  • NRGl multiple sclerosis, systemic lupus erythematosus or related diseases
  • additional susceptibility alterations can be identified within said gene or polypeptide, e.g., following the methodology disclosed in the examples.
  • the presence of an alteration in the NRGl gene may be detected by any technique known per se to the skilled artisan (reviewed by Kwok et al., 2003), including sequencing, pyrosequencing, selective hybridisation, selective amplification and/or mass spectrometry including matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004).
  • the alteration is detected by selective nucleic acid amplification using one or several specific primers.
  • the alteration is detected by selective hybridization using one or several specific probes.
  • Further techniques include gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping; rolling circle amplification and Invader assays as well as DNA chip-based microarray and mass spectrometry genotyping technologies (Shi et al., 2001).
  • gel electrophoresis-based genotyping methods such as PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing
  • fluorescent dye-based genotyping technologies such as oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homo
  • RNA expression of altered genes can be quantified by methods known in the art such as subtractive hybridisation, quantitative PCR, TaqMan, differential display reverse transcription PCR, serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips.
  • Particular methods include allele-specific oligonucleotide (ASO), allele-specific amplification, fluorescent in situ hybridization (FISH) Southern and Northern blot, and clamped denaturing gel electrophoresis.
  • Protein expression analysis methods include 2-dimensional gel- electrophoresis, mass spectrometry and antibody microarrays (Freeman et al., 2004 and Zhu et al., 2003).
  • Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA). These techniques can be performed using commercially available reagents and protocols.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • a preferred technique is allele-specific PCR.
  • Nucleic acid primers useful for amplifying sequences from the NRGl gene are able to specifically hybridize with a portion of the NRGl gene that either flanks or overlaps with a susceptibility alteration.
  • the primer sequence overlaps with the alteration when said alteration is contained within the sequence of the NRGl gene to which the primer hybridizes.
  • the primer sequence flanks the alteration when the primer hybridizes with a portion of the NRGl gene that is preferably located at a distance below 300 bp of said alteration, even more preferably below 250, 200, 150, 100, 50, 40, 30 or 20 bp from said alteration.
  • the primer hybridizes with a portion of the NRGl gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
  • the methods involve the use of a nucleic acid probe specific for a NRGl or altered NRGl gene, followed by the detection of the presence of a hybrid.
  • the probe may be used in suspension or immobilized on a substrate or support.
  • the probe is typically labelled to facilitate detection of hybrids.
  • Treatments include, for instance, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc.
  • the nucleic acids and/or polypeptides may be pre- purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. Considering the high sensitivity of the claimed methods, very few amounts of sample are sufficient to perform the assay.
  • the finding of an altered NRGl gene or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in the subject.
  • a related disorder in the subject.
  • one only of the above-disclosed susceptibility alterations is assessed, or several of them, in combination(s).
  • kits for the identification of a genetic polymorphism pattern at the NRGl gene associated with increased risk of the presence of or predisposition to multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder in a subject comprising:
  • (b) means for determining a genetic polymorphism pattern for the NRGl gene.
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads.
  • These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a NRGl gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, and further preferably a compound that can be used for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder, or as a lead to develop drugs for treating multiple sclerosis, systemic lupus erythematosus, psoriasis or a related disorder.
  • a “biologically active” compound preferably is a compound that modulates the activity of NRGl.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a NRGl gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • active compounds particularly compounds active on multiple sclerosis, systemic lupus erythematosus, psoriasis or related disorders
  • the modulator is a NRGl fusion protein.
  • a fusion protein of NRGl may e.g. comprise an immunoglobulin fusion, i.e. a fused protein comprising all or part of a NRGl protein, which is fused to all or a portion of an immunoglobulin.
  • Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737, for example. The person skilled in the art will appreciate that the resulting fusion protein of the invention substantially retains the biological activity of NRGl, which can be measured in in vitro assays.
  • the modulator is an inhibitor or antagonist of a NRGl polypeptide.
  • a further object of this invention resides in a pharmaceutical composition
  • a pharmaceutical composition comprising a functional NRGl polypeptide or a nucleic acid encoding a NRGl polypeptide or a vector encoding the same, and a pharmaceutically acceptable diluent or carrier.
  • Selected for the SLE study were individuals who fulfilled at least 4 of the 11 ACR criteria.
  • Autoprimer.com automatically optimizes the grouping of the alterations by extension mix and appends tag sequences to the 5' ends of the SNP-IT primers, which are complementary to the tags immobilized on the microarray plate.
  • Affymetrix genotyping technology Use of Affymetrix genotyping technology have been described in Kennedy, G.C., et al. Nature Biotechnology 21, 1233-1237, 2003 (Large-scale genotyping of complex DNA). ; Liu, W.M., et al. Bioinformatics 19, 2397-2403, 2003. (Algorithms for Large Scale Genotyping Microarrays) ;Matsuzaki, H., et al. Genome Research 3, 414-25, 2004. (Parallel Genotyping of over 10,000 SNPs using a One Primer Assay on a High Density Oligonucleotide Array) ;Paez, J.G., et al. Nucleic Acids Research 32(9), 2004.
  • Hybridization signals are generated in a three step signal amplification process: lO ⁇ g/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5 ⁇ g/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C.
  • SAPE streptavidin R-phycoerythrin conjugate
  • the SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (10OmM MES, IM [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000 scanners with
  • Genomic Control (Devlin andRoeder 1999): given that in the presence of population substructure, the standard chi-square statistic is inflated by a multiplicative factor, which is proportional to the degree of stratification, we can estimate and incorporate this multiplicative factor (lambda) into the disease - susceptibility alteration association tests (by rescaling the chi-square statistic) to correct for background population differences.

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Abstract

La présente invention se rapporte en général à des méthodes et à des compositions permettant de détecter ou de traiter des troubles inflammatoires, tels que la sclérose en plaques, le lupus érythémateux disséminé ou le psoriasis. L'invention concerne plus particulièrement l'identification de gènes humains qui peuvent servir au diagnostic, à la prévention et au traitement de la sclérose en plaques, du lupus érythémateux disséminé, du psoriasis et de troubles associés, ainsi qu'au criblage de médicaments thérapeutiquement actifs. L'invention a également trait à des polymorphismes, à des variants d'épissage ou à des allèles spécifiques du gène NRG1 qui sont associés à la sclérose en plaques, au lupus érythémateux disséminé et au psoriasis, ainsi qu'à des outils et à des kits de diagnostic basés sur lesdits polymorphismes. L'invention peut servir au diagnostic de la sclérose en plaques, du lupus érythémateux disséminé, du psoriasis et de troubles associés, au diagnostic d'une prédisposition auxdites maladies, ainsi qu'à la détection, à la prévention, et/ou au traitement desdites maladies.
EP06725045A 2005-03-15 2006-03-14 Compositions et methodes destinees a traiter et a diagnostiquer des troubles inflammatoires Withdrawn EP1863927A2 (fr)

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EP06725045A EP1863927A2 (fr) 2005-03-15 2006-03-14 Compositions et methodes destinees a traiter et a diagnostiquer des troubles inflammatoires
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ES2661249T3 (es) * 2007-05-21 2018-03-28 Genentech, Inc. Métodos y composiciones para identificar y tratar el lupus
WO2009137837A2 (fr) * 2008-05-09 2009-11-12 The Regents Of The University Of California Signalisation de neuréguline/erbb et intégrine
WO2010033951A2 (fr) * 2008-09-19 2010-03-25 University Of Utah Research Foundation Procédé d'identification et de prédiction de la sclérose en plaques et de la réponse à la thérapie
WO2010094525A1 (fr) * 2009-01-16 2010-08-26 Merck Serono S.A. Marqueurs génétiques pour le diagnostic de formes progressives primaires de la sclérose en plaques
EP4347892A2 (fr) * 2021-06-03 2024-04-10 Merus N.V. Essais de biopsie liquide pour détecter des polynucléotides de fusion nrg1

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US5770567A (en) * 1994-11-14 1998-06-23 Genentech, Inc. Sensory and motor neuron derived factor (SMDF)
CA2306228A1 (fr) * 1997-10-14 1999-04-22 Cambridge Neuroscience, Inc. Methode therapeutiques comportant l'utilisation d'une neureguline
AU7873200A (en) * 1999-10-08 2001-04-23 Uab Research Foundation Smdf and ggf neuregulin splice variant isoforms and uses thereof
JP2004513607A (ja) * 2000-02-28 2004-05-13 デコード　ジェネティクス　イーエッチエフ． ヒト精神分裂病遺伝子
AU2002337671A1 (en) * 2001-08-07 2003-02-24 Genaissance Pharmaceuticals, Inc Polymorphisms associated with multiple sclerosis
AUPS271902A0 (en) * 2002-05-31 2002-06-20 Griffith University Gene expression and multiple sclerosis

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