[go: up one dir, main page]

WO2007071437A2 - Compositions et méthodes pour traiter des troubles inflammatoires - Google Patents

Compositions et méthodes pour traiter des troubles inflammatoires Download PDF

Info

Publication number
WO2007071437A2
WO2007071437A2 PCT/EP2006/012458 EP2006012458W WO2007071437A2 WO 2007071437 A2 WO2007071437 A2 WO 2007071437A2 EP 2006012458 W EP2006012458 W EP 2006012458W WO 2007071437 A2 WO2007071437 A2 WO 2007071437A2
Authority
WO
WIPO (PCT)
Prior art keywords
pap
paplb
seq
polypeptide
gene
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.)
Ceased
Application number
PCT/EP2006/012458
Other languages
English (en)
Other versions
WO2007071437A3 (fr
Inventor
Emmanuelle Laurine
Ilya Chumakov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ares Trading SA
Original Assignee
Ares Trading SA
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ares Trading SA filed Critical Ares Trading SA
Publication of WO2007071437A2 publication Critical patent/WO2007071437A2/fr
Publication of WO2007071437A3 publication Critical patent/WO2007071437A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

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).
  • the present invention more particularly relates to the human PAP (pancreatitis associated protein) and PAPlB gene, which can be used for the diagnosis, prevention and treatment of multiple sclerosis 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 PAP or PAPlB gene that are related to inflammatory disorders, such as multiple sclerosis, 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 and related disorders.
  • MS Multiple sclerosis
  • MS may result in the accumulation of various neurological disabilities.
  • Clinical disability in MS is presumed to be a result of repeated inflammatory injury with subsequent loss of myelin and axons, leading to tissue atrophy.
  • MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment. Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur. Approximately half of the patients with MS may experience cognitive impairment or depression. MS is now considered to be a multi-phasic disease and periods of clinical quiescence (remissions) occur between exacerbations. Remissions vary in length and may last several years but are infrequently permanent.
  • RR relapsing-remitting
  • SP secondary progressive
  • PP primary progressive
  • PR progressive relapsing
  • More than 80% of patients with MS will initially display a RR course with clinical exacerbation of neurological symptoms, followed by a recovery that may or may not be complete ⁇ Lublin and Reingold, Neurology, 1996, 46:907-911).
  • MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction.
  • CSF cerebrospinal fluid
  • MRI magnetic resonance imaging
  • Molecules currently used for the treatment of multiple sclerosis may have side effects and may act only against the symptoms of the disease. Consequently, there is a strong need for new molecules without or with less associated side effects that are directed against novel targets. Therefore, there is a need to identify proteins involved in the diseases, thereby providing new targets allowing new screenings for drugs, resulting in new drugs that are efficient in treatment of these serious inflammatory diseases and related disorders.
  • the present invention now discloses novel approaches to the diagnosis and treatment of multiple sclerosis as well as for the screening of therapeutically active drugs.
  • the invention more specifically demonstrates that alterations in the PAP gene are associated with the development of multiple sclerosis.
  • PAP, and altered forms of PAP in particular, represent novel targets for therapeutic intervention against MS and related pathologies.
  • PAPlB is co-regulated with PAP and therefore it may also be regarded as associated with the development of multiple sclerosis.
  • a first aspect of this invention relates to methods of treating multiple sclerosis or related disorders in a subject through a modulation of PAP or PAPlB gene or polypeptide expression or activity, preferably through an activation or restoration thereof.
  • Such treatments use, for instance, a PAP or PAPlB polypeptide, a PAP or PAPlB DNA sequence (including antisense sequences, RNAi), antibodies against PAP or PAPlB polypeptides, ligands of PAP or PAPlB or drugs that modulate, preferably mimic or stimulate, PAP or PAPlB expression or activity.
  • the invention particularly relates to methods of treating individuals having disease-associated alleles of the PAP or PAPlB gene such as further described herein, in particular in Tables 2a and 2b.
  • a further aspect of this invention thus resides in the use of a PAP or PAPlB gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against multiple sclerosis and related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising determining the ability of a compound to bind a PAP or PAPlB gene or polypeptide, or a fragment thereof, particularly of an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of multiple sclerosis or related disorders, comprising testing for modulation of the activity of a PAP or PAPlB gene or polypeptide, particularly an allele of said gene or polypeptide that is associated with multiple sclerosis or a related disorder, or a fragment thereof.
  • Another aspect of this invention resides in a method of assessing the presence of or predisposition to multiple sclerosis or a related disorder in a subject, comprising determining (in vitro or ex vivo) the presence of an alteration (e.g., a susceptibility mutation or allele) in a PAP or PAPlB 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 or a related disorder in said subject.
  • an alteration e.g., a susceptibility mutation or allele
  • a further aspect of this invention relates to the use of a modulator of a PAP or PAPlB gene or polypeptide, preferably an agonist thereof, for the preparation of a medicament for treating or preventing multiple sclerosis or a related disorder in a subject, as well as to corresponding methods of treatment.
  • a further aspect of this invention relates to the screening of alteration(s) associated with multiple sclerosis or related disorders in the PAP or PAPlB gene locus in patients. Such screenings are useful for diagnosing the presence, risk or predisposition to multiple sclerosis and related disorders, and/or for assessing the efficacy of a treatment of such disorders.
  • a further aspect of this invention includes nucleic acid probes and primers that allow specific detection of susceptibility alterations in a PAP or PAPlB gene or RNA through selective hybridization or amplification.
  • the invention also encompasses particular nucleic acids, vectors and recombinant cells, as well as kits or solid phase bound nucleic acids or proteins such as DNA or protein arrays or chips suitable for implementing the above detection, screening or treatment methods.
  • the invention also discloses and encompasses susceptibility alterations in the PAP or PAPlB nucleic acids and polypeptides that are associated with multiple sclerosis and related disorders. Examples of such susceptibility alterations are more particularly selected from the SNPs as listed in Table 2a and 2b.
  • the invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of multiple sclerosis and related disorders in any mammalian subjects, particularly human patients.
  • the invention relates to antibodies, which specifically bind to the PAP or PAP 1 B polypeptide.
  • Figure 1 Genomic organization of PAPlB (plus direction, chromosome 2 at location 79,164,481-79,167,279), REGlB (minus direction, chromosome 2 at location 79,223,806- 79,226,774.), REGlA (plus direction, chromosome 2 at location 79,259,239-79,262,200), PAP (minus direction, chromosome 2 at location 79,295,788-79,298,534) and REG4 (plus direction, chromosome 1 at location 120,048,683-120,066,325).
  • Figure 2 Alignment of three known PAP splice variants with the PAP protein sequence. All three PAP splice variants encode the same PAP protein.
  • PAPlB splice variants 1 and 2 both encode isoform 1 of PAPlB protein whereas PAPlB splice variant 3 encodes isoform 3 of PAPlB protein.
  • FIG. 3 PAP, PAPlB isoform 1 (PAPlB-il) and PAPlB isoform 3 (PAPlB-i3) multiple sequence alignment. Signal peptides are underlined.
  • Figure 4 PAP activity on MBP production in maturating mixed spinal cord cultures expressed as a ratio between MBP and total protein amount (A) and as the amount of MBP per well (B). Effect of PAP on total protein amount (C).
  • the present invention stems from association studies conducted on different MS populations, using a number of random alterations. The results of these studies show that the PAP gene is strongly associated with multiple sclerosis and that new and validated (biallelic) susceptibility alterations located in said gene are associated with multiple sclerosis. It has also been found that PAP and PAPlB genes are co-regulated. PAPlB may therefore also be regarded as associated with multiple sclerosis.
  • results of an in vitro model show that PAP is able to induce myelin production in vitro and an in vivo (rat EAE) model further strengthens the functional link between PAP and MS.
  • the present invention thus provides novel approaches to the prevention and treatment of multiple sclerosis or related disorders in a subject.
  • the invention further provides novel approaches to the detection, diagnosis and monitoring of multiple sclerosis or related disorders in a subject, as well as for genotyping of inflammatory patients, in particular patients having MS.
  • the invention further provides novel means and methods to identify compounds useful in the treatment of multiple sclerosis and related disorders.
  • multiple 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).
  • the term "inflammatory disorder” includes in particular demyelinating inflammatory CNS disorders, such as for example MS and related disorders.
  • Disorders related to MS include progressive multifocal leukoencephalopathy (PML), acute disseminated encephalomyelitis (ADEM), acute demyelinating polyneuropathy (Guillain Barre syndrome), chronic inflammatory demyelinating neuropathy, Marchivafa-Bignami disease, central pontine myelinolysis, Devic syndrome, BaIo disease, HFV- or HTLV- myelopathy or a secondary demyelinating disorder such as CNS lupus erythematodes, polyarteritis nodosa, Sjogren syndrome, sarcoidosis or isolated cerebral vasulitis.
  • PML progressive multifocal leukoencephalopathy
  • ADAM acute disseminated encephalomyelitis
  • UDM acute demyelinating polyneuropathy
  • Chronic inflammatory demyelinating neuropathy
  • PAP designates the human PAP gene, as well as variants, analogs and fragments thereof. Synonyms of human PAP are Pancreatitis-associated protein, Pancreatitis-associated protein 1 (PAPl), Regenerating islet-derived protein 3 alpha precursor (Reg Ill-alpha) and Hepatocarcinoma-Intestine- Pancreas/ Pancreatic associated protein (HIP/PAP).
  • PAPlB designates the human PAPlB gene, as well as variants, analogs and fragments thereof. Synonyms of human PAPlB are Pancreatitis-associated protein IB, Regenerating islet-derived protein 3 gamma precursor (Reg Ill-gamma) and PAP-2.
  • PAP belongs to the REG family which contains 19 members only present in mammals. In human, this family is composed by 5 genes, 4 of them form a cluster on chromosome 2 [REG IA, REG IB, PAP(REG3A), PAP1B(REG3G)] the fifth being located on chromosome 1 (REG 4), see Figure 1 (Laurine E. et al., Biochim Biophys Acta. 2005 Mar 10;1727(3):177-87).
  • PAP encodes a secreted protein of 175 amino acids, including a N-terminal signal peptide of 26 amino acids.
  • PAP belongs to the C-type lectin superfamily according to its sequence and its ability to bind lactose (Christa L. et al., FEBS Lett. 1994 Jan 3;337(1):114-8).
  • PAP and PAPlB gene or polypeptide are available in the literature. They are described in Figure 2. Three splice variants of PAP are known. Their nucleic acid sequences are defined herein as SEQ ID NO. 1, SEQ ID. NO.2 and SEQ ID NO.3. They all code for the same protein.
  • SEQ ID NO. 4 The full length PAP protein including signal sequence is defined herein as SEQ ID NO. 4 and the mature secreted form of PAP protein is defined herein as SEQ ID NO. 5.
  • Splice variants of PAPlB are known. Their nucleic acid sequences are defined herein as SEQ ID NO. 6, SEQ ID. NO.7 and SEQ ID NO.10. Splice variants with SEQ ID NO. 6 and SEQ ID NO. 7 code for the same protein (isoform 1).
  • This full length PAPlB protein isoform 1 including signal sequence is defined herein as SEQ ID NO. 8 and the mature secreted form of this PAPlB protein isoform 1 is defined herein as SEQ ID NO. 9.
  • Splice variant with SEQ ID NO. 10 codes for PAPlB protein isoform 3.
  • the full length PAPlB protein isoform 3 including signal sequence is defined herein as SEQ ID NO. 11 and the mature secreted form of this PAPlB protein isoform 3 is defined herein as SEQ ID NO. 12.
  • PAP and PAPlB share 85.1% amino acid sequence identity.
  • PAP and PAPlB are both up regulated in injured nerve (Namikawa et al., BBRC. 2005 Apr 332:126-134), in EAE rat model (Example 2) and during colonic bacterial colonization of germ free mice (Keilbaugh et al., Gut. 2005 May 54: 623-629) suggesting that they may be co-regulated.
  • PAP is formed by a N-terminal undecapeptide linked to a C- terminal carbohydrate recognition domain (CRD) of 138 amino acids.
  • CRD carbohydrate recognition domain
  • the junction between these 2 domains is susceptible to trypsic digestion leading to oligomerization of CRD domain in fibrillar aggregates (Graf R. et al., J Biol Chem. 2001 Jun 15;276(24):21028-38).
  • PAP expression is time and tissue specific. In brain, PAP is expressed during development at late gestation stages in nervous system, its expression being restricted in specific areas containing sensory or motoneurons. In adults no expression is detected in brain (Lasserre C. et al., Am J Pathol. 1999 May;154(5):1601-1610; Laurine E. et al., Biochim Biophys Acta. 2005 Mar 10;1727(3):177-87 ). Expression in pancreas and small intestine begins at late stages of gestation and becomes strong in adults (Lasserre C. et al., Cancer Res. 1992 Sep l5;52(18):5089-95).
  • PAP expression is altered in several human diseases. PAP is overexpressed in inflammatory diseases such as Acute and Chronic Pancreatitis (Orelle B. et al., J Clin Invest. 1992 Dec;90(6):2284-91) and Inflammatory Bowel Disease (Gironella M. et al., Gut. 2005 Sep;54(9): 1244-53). PAP expression is also increased in Type I diabetes and in Non Obese Diabetes mice model (Gurr W. et al., Diabetes. 2002 Feb;51(2):339-46). PAP is up regulated in carcinogenesis in liver, pancreas, stomach and colon. In addition, PAP expression is associated with Alzheimer's disease. High levels of PAP were detected in cerebrospinal fluid since early stages of the disease and PAP formed deposits in patient's brain (Duplan et al., Neurobiol. Aging, 2001 Jan-Feb; 22(1), 79-88).
  • PAP has been described as a neurotrophic factor which induces neuronal survival and Schwann cell proliferation in vitro (Livesey FJ et al., Nature. 1997 Dec 1 1 ;390(6660):614- 25; Namikawa K. et al., Biochem Biophys Res Commun. 2005 Jun 24;332(l):126-34.; EP 903147 A).
  • mice developing peripherical neuropathy treated with peptide fragment of PAP showed enhanced regeneration and nerve function improvement (Tarn J at al., Biochem Biophys Res Commun. 2002 Mar l;291(3):649-54; Tarn J et al., FASEB J.
  • WO 02/070551 discloses that a small portion of INGAP is sufficient to stimulate repair and/or regeneration of peripheral nervous system. The teaching of WO 02/070551 is herein included by reference.
  • 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 PAP gene according to this invention has the sequence defined in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
  • the PAPlB gene according to this invention has the sequence as defined in SEQ ID NO. 6, SEQ ID NO.7 or SEQ ID NO.10.
  • the present invention also encompasses fragments of the PAP or PAPlB 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 or 175 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 PAP or PAPlB 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 PAP or PAPlB 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 al., Current Protocols in Molecular Biology, supra, Interscience, N. Y., ⁇ 6.3 and 6.4 (1987, 1992), and Sambrook et al. (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 PAP or PAPlB 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, 150, 175 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 PAP polypeptide according to this invention has the sequence defined in SEQ ID NO. 4.
  • the PAP polypeptide according to this invention has the sequence of the mature peptide as defined in SEQ ID NO. 5.
  • the PAPlB polypeptide according to this invention has the sequence as defined in SEQ ID NO. 8 or SEQ ID NO. 11.
  • the PAPlB polypeptide according to this invention has the sequence of the mature peptide as defined in SEQ ID NO. 9 or SEQ ID NO. 12.
  • the present invention also encompasses peptide sequences, which have at least 40% identity with the sequence of SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO. 11 or SEQ ID NO. 12. More preferably, they have at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity thereto.
  • the present invention also encompasses fragments of PAP or PAPlB or mutants thereof.
  • the fragments may comprise at least 15, 25, 50, 75, 100, 150 or 175 contiguous amino acids of PAP or PAPlB.
  • the mutants may have some amino acid exchanges compared to the human PAP or PAPlB sequence fragments.
  • a preferred fragment of PAP has the amino acid sequence as defined in SEQ ID NO: 17. It is the human homologue of the pentadecapeptide INGAP fragment disclosed in WO 02/070551.
  • a preferred mutant of the fragment has the amino acid sequence as defined in SEQ ID NO: 18. It corresponds to pentadecapeptide INGAP fragment disclosed in WO 02/070551.
  • Fusion proteins are useful for generating antibodies against a PAP or PAPlB polypeptide and for use in various assay systems. For example, fusion proteins can be used to identify proteins, which interact with portions of a PAP or PAPlB polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a PAP or PAPlB polypeptide fusion protein comprises two polypeptide segments operatively linked together by means of a peptide bond.
  • the first polypeptide segment comprises at least 15, 25, 50, 75, 100, 150 or 175 contiguous amino acids of PAP or PAPlB.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include beta- galactosidase, beta-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • 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.
  • 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) BP 16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the PAP or PAPlB polypeptide- encoding sequence and the heterologous protein sequence, so that the PAP or PAPlB polypeptide can be cleaved and purified away from the heterologous moiety.
  • fusion protein In the context of a fusion protein, the expression "operably linked” indicates that the PAP or PAPlB polypeptide and additional amino acid sequences are associated through peptide linkage(s), either directly or via spacer residues (e.g., a linker).
  • 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 PAP or PAPlB 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 0 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 PAP or PAPlB 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.
  • 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. It is preferred that the antibodies according to the present invention exhibit binding affinity (Ka) to the target polypeptide or epitope of 10 6 M “1 or greater, preferably 10 7 M "1 or greater, more preferably 10 8 M "1 or greater and most preferably 10 9 M "1 or greater.
  • Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • an immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the polypeptide of SEQ ID NO 4, 5, 8, 9, 11 or 12 or a variant as described hereabove or a fusion protein thereof.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separared.
  • the antibodies may, alternatively, be monoclonal antibodies.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent (the immunizing agent will typically include the polypeptide of SEQ ID NO: 4, 5, 8, 9, 11 or 12 or a variant as described hereabove or a fusion protein thereof) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the immunizing peptide.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purificationproceduressuch as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein.
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 [1991] and Marks et al., J.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • 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 of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab 1 , F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Methods for humanizing non-human antibodies are well known in the art. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)).
  • the techniques of Cole et al., and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
  • human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to heterologous moieties, such as cytotoxic agents, labels, drugs or other therapeutic agents, covalently bound or not, either directly or through the use of coupling agents or linkers.
  • heterologous moieties such as cytotoxic agents, labels, drugs or other therapeutic agents, covalently bound or not, either directly or through the use of coupling agents or linkers.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • antibodies or antibody fragments of the present invention can be PEGylated using methods in the art and described herein.
  • the antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • antibody fragments which comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng., 8(10): 1057-1062 [1995]); single-chain antibody molecules; monobodies; camelized monobodies; domain antibodies and multispecific antibodies formed from antibody fragments.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • Single-chain antibody molecules are fragments of an antibody comprising the VH and VL domains of said antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the single-chain antibody molecule to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • a monobody can bind to an antigen in the absence of light chains and typically has three CDR regions designated CDRHl, CDRH2 and CDRH3.
  • a heavy chain IgG monobody has two heavy chain antigen binding molecules connected by a disulfide bond.
  • the heavy chain variable domain comprises one or more CDR regions, preferably a CDRH3 region.
  • a “camelized monobody” refers to a monobody or antigen binding portion thereof obtained from a source animal of the camelid family, including animals with feet with two toes and leathery soles. Animals in the camelid family include camels, llamas, and alpacas. It has been reported that camels (Camelus dromedaries and Camelus bactrianus) often lack variable light chain domains when IgG-like material from their serum is analyzed, suggesting that sufficient antibody specificity and affinity can be derived form VH domains (three CDR loops) alone.
  • Single domain antibodies also called domain antibodies or dAbs, are the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies.
  • Domain antibodies have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody.
  • domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems.
  • many domain antibodies are highly stable and retain activity even after being subjected to harsh conditions, such as freeze-drying or heat denaturation which makes them amenable to a wide range of pharmaceutical formulation conditions and manufacture processes. DETECTION AND DIAGNOSIS
  • the present invention provides novel means and methodologies for detecting or diagnosing multiple sclerosis 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 or a related disorder in a subject, the method comprising detecting the presence of an alteration in a PAP or PAPlB 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 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 or a related disorder, the methods comprising detecting the presence of an alteration in a PAP or PAPlB gene or polypeptide in a sample from the subject, the presence of such an alteration being indicative of a responder subject.
  • Medications for MS within the meaning of the present invention include the four FDA approved immunomodulating agents for RRMS: three beta interferons (Betaseron®, Berlex; Avonex®, Biogen; Rebif®, Serono) and Glatimarer Acetate (Copaxone®, Amgen). Medications for MS within the meaning of the present invention also include the FDA approved immunosuppressing drug for worsening MS, Mitoxantrone (Novantrone®, Amgen).
  • 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).
  • the use of interferons of human origin is also preferred in accordance with the present invention.
  • 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.
  • the dosing of IFN- ⁇ in the treatment of relapsing-remitting MS according to the invention depends on the type of IFN- ⁇ used.
  • 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.
  • IFN when IFN is recombinant IFN- ⁇ Ia, produced in Chinese Hamster Ovary cells (CHO cells), commercially available under the trademark Rebif, it may preferably be administered sub-cutaneously three times a week (TIW) at a dosage of 22 to 44 g or 6 MIU to 12 MIU per person.
  • TIW sub-cutaneously three times a week
  • the alteration ("susceptibility alteration") in a PAP or PAPlB gene or polypeptide may be any nucleotide or amino acid alteration associated to multiple sclerosis or a related disease.
  • a susceptibility alteration in the PAP or PAPlB 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 PAP or PAPlB 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 PAP or PAPlB 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).
  • the present invention now discloses several susceptibility alterations in the PAP gene, which are associated with multiple sclerosis. These mutations are reported in table 2a and table 2b. Furthermore, results of an in vitro model show hat PAP is able to induce myelin production in vitro, and an in vivo (rat EAE) model further strengthens the functional association between PAP and MS. In summary, the association results of the single biallelic alteration frequency analysis, the myelin production experiment and the EAE model the further experiments show that the PAP gene is associated with multiple sclerosis. It has also been surprisingly found that PAPlB is coregulated with PAP and may also be regarded as associated with multiple sclerosis.
  • Preferred genetic alterations linked to MS are disclosed in table 2a below. Most preferred genetic alterations linked to MS are disclosed in table 2b below.
  • DNA strand -1 reverse strand
  • +1 forward strand
  • Base 1 base number 79295788 of chromosome 2 to base number 79298534
  • Ch. Allele The Chosen Allele is the allele which frequency is increased within cases as compared to controls K)
  • An object of the present invention comprises the detection of the presence of a susceptibility alteration as disclosed in Tables 2a in the PAP gene or RNA sequence of a subject, more particularly the detection of at least one alteration as disclosed in Table 2b or any combination thereof.
  • a preferred object of this invention is a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject, the method comprising detecting the presence or absence of the associated susceptibility alteration according to table 2a or 2b in a sample from the subject, the presence of the associated susceptibility alteration being indicative of the presence of or predisposition to multiple sclerosis or a related disorder in said subject.
  • the presence of an alteration in the PAP 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).
  • Sequencing can be carried out using techniques well known in the art, using automatic sequencers.
  • the sequencing may be performed on the complete gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
  • 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 PAP or PAPlB gene are able to specifically hybridize with a portion of the PAP or PAPlB 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 PAP or PAPlB gene to which the primer hybridizes.
  • the primer sequence flanks the alteration when the primer hybridizes with a portion of the PAP 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 PAP gene that is at 5, 4, 3, 2, 1 bp distance or immediately adjacent to said alteration.
  • the invention also relates to the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder.
  • the methods involve the use of a nucleic acid probe specific for a PAP or altered PAP 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.
  • a specific object of this invention is a nucleic acid probe complementary to and specific for a region of a PAP gene that carries an alteration as described in Table 2a or 2b.
  • the probes of the present invention are, more preferably, capable of discriminating between an altered and non-altered PAP gene sequence, i.e., they specifically hybridise to a PAP gene carrying a particular alteration as described above, and essentially do not hybridise under the same hybridization conditions or with the same stability to a PAP gene lacking said alteration.
  • the invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to multiple sclerosis or a related disorder in a subject or in a method of assessing the response of a subject to a treatment of multiple sclerosis or a related disorder.
  • the detection methods can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. They are typically performed on a sample from the subject, such as any biological sample containing nucleic acids or polypeptides. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc.
  • the sample may be collected according to conventional techniques and used directly for diagnosis or stored. In particular, they may be obtained by non-invasive methods, such as from tissue collections.
  • the sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing.
  • 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 sample is typically contacted with probes or primers as disclosed above.
  • Such contacting may be performed in any suitable device, such as a plate, tube, well, glass, etc.
  • the contacting may be performed on a substrate coated with said specific reagents, such as a nucleic acid array.
  • the substrate may be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids of the sample.
  • the finding of an altered PAP gene or polypeptide in the sample is indicative of the presence, predisposition or stage of progression of multiple sclerosis or 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 PAP gene associated with increased risk of the presence of or predisposition to multiple sclerosis or a related disorder in a subject comprising:
  • (b) means for determining a genetic polymorphism pattern for the PAP 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.
  • a particular object of this invention resides in the use of a PAP or PAPlB polypeptide as a target for screening candidate drugs for treating or preventing multiple sclerosis or a related disorder.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a PAP or PAPlB gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a PAP or PAPlB polypeptide with a candidate compound, and selecting compounds that bind said PAP or PAPlB polypeptide and/or that modulate the activity of the PAP or PAPlB 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 or a related disorder, or as a lead to develop drugs for treating multiple sclerosis or a related disorder.
  • a “biologically active” compound preferably is a compound that modulates the activity of PAP or PAPlB.
  • the above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of multiple sclerosis or a related disorder, such as an animal model.
  • Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to multiple sclerosis or a related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
  • Modulation of activity includes, without limitation, stimulation of the surface expression of the PAP or PAPlB receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a PAP or PAPlB polypeptide) or any cell that expresses an endogenous PAP or PAPlB polypeptide.
  • examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Preferred selected compounds are agonists of PAP or PAPlB, i.e., compounds that can bind to PAP or PAPlB receptor and mimic the activity of an endogenous ligand thereof.
  • the screening assays of the present invention use, either alone or in addition to another PAP or PAPlB sequence, an altered PAP or PAPlB gene or polypeptide, particularly a PAP gene or polypeptide having an alteration as listed in Table 2a or 2b.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a PAP or PAPlB polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said p PAP or PAPlB polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a PAP or PAPlB gene according to the present invention and determining the ability of said test compound to modulate the expression of said PAP or PAPlB gene, preferably to stimulate expression thereof.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis 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 PAP or PAPlB gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • this invention relates to the use of a PAP or PAPlB polypeptide or fragment thereof, whereby the fragment is preferably a PAP or PAPlB gene-specific fragment, for isolating or generating an agonist or stimulator of the PAP or PAPlB polypeptide for the treatment of multiple sclerosis or a related disorder, wherein said agonist or stimulator is selected from the group consisting of: 1. a specific antibody or fragment thereof including a) a chimeric, b) a humanized or c) a fully human antibody as well as 2. a bispecific or multispecific antibody, 3. a single chain (e.g. scFv) or
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. tri
  • the present invention now discloses novel approaches to the treatment of multiple sclerosis and related disorders by modulating the activity or expression of a PAP or PAPlB gene or polypeptide. More particularly, the present invention provides the first evidence of a genetic correlation between said PAP gene and said diseases in human subjects.
  • the present invention provides the first in vitro and in vivo models showing the functional association of PAP or PAPlB with demyelinating diseases. This allows the design of novel therapeutic approaches based on a modulation, preferably a stimulation or increase of a PAP or PAPlB activity.
  • a particular object of this invention resides in the use of a PAP or PAPlB polypeptide, or a nucleic acid encoding the same, for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • a preferred embodiment resides in the use of a PAP nucleic acid of SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 or in the use of a PAPlB nucleic acid of SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 10 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • Another preferred embodiment resides in the use of a PAP polypeptide of SEQ ID NO. 4 or SEQ ID NO. 5 or in the use of a PAPlB polypeptide of SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO. 1 1 or SEQ ID NO. 12 for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • a further object of this invention resides in the use of a modulator of PAP or PAPlB for the manufacture of a pharmaceutical composition for treating or preventing multiple sclerosis or a related disorder in a subject.
  • the modulator is an agonist or activator of a PAP or PAPlB polypeptide.
  • the modulator is a PAP or PAPlB fusion protein.
  • a fusion protein of PAP or PAPlB may e.g. comprise an immunoglobulin fusion, i.e. a fused protein comprising all or part of a PAP or PAPlB protein, which is fused to all or a portion of an immunoglobulin.
  • fusion protein of the invention substantially retains the biological activity of PAP or PAPlB, which can be measured in in vitro assays.
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or for example a 13 -amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu- Val-Leu-Gly-Gly-Gln-Phe-Met introduced between the PAP or PAPlB sequence and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • PAP or PAPlB is fused to the constant region of an Ig molecule, e.g. an Fc portion of an Immunoglobulin.
  • Ig molecule e.g. an Fc portion of an Immunoglobulin.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains, optionally with the hinge region of human IgGl, for example.
  • the Fc part may e.g. be mutated in order to prevent unwanted activities, such as complement binding, binding to Fc receptors, or the like.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG 2 or IgG 4 , or other Ig classes, like IgM or IgA, for example.
  • Fusion proteins may be monomelic or multimeric, hetero- or homomultimeric. Further fusion proteins of PAP or PAPlB may be prepared by fusing domains isolated from other proteins allowing the formation of dimers, trimers, etc. Examples for protein sequences allowing the multimerization of the polypeptides of the Invention are domains isolated from proteins such as hCG (WO 97/30161), collagen X (WO 04/33486), C4BP (WO 04/20639), Erb proteins (WO 98/02540), or coiled coil peptides (WO 01/00814). In one embodiment the PAP or PAPlB is fused to the C-terminal peptide of the beta-chain of hCG.
  • the modulator is a fusion protein between the Fc region of an antibody and a PAP polypeptide defined by SEQ ID NO. 4 or SEQ ID NO. 5 or a PAPlB protein defined by SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO. 11 or SEQ ID NO. 12.
  • the proteins of the Invention can be in the form of an active conjugate or complex with a molecule chosen amongst radioactive labels, biotin, fluorescent labels, cytotoxic agents, and drug delivery agents.
  • Useful conjugates or complexes can be generated, using molecules and methods known in the art, for various reasons, for example for allowing the detection of the interaction with other proteins (radioactive or fluorescent labels, biotin), therapeutic efficacy (cytotoxic agents), or improving the agents in terms of drug delivery efficacy, such as polyethylene glycol and other natural or synthetic polymers.
  • the present invention contemplates chemically modified polypeptides and proteins as disclosed herein, in which the polypeptide or the protein is linked with a polymer.
  • the polymer is water soluble so that the conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled.
  • An example of a reactive aldehyde is polyethylene glycol propionaldehyde, ormono- (Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, U. S. Patent No. 5,252, 714).
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce the conjugates.
  • the conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties.
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono- (Cl-ClO) alkoxy-PEG, aryloxy- PEG, poly- (N-vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.
  • PEG polyvinyl alcohol
  • dextran cellulose
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • a conjugate can also comprise a mixture of such water- soluble polymers.
  • PEGylation can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316).
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, U. S. Patent No. 5,382, 657).
  • the PEG may be linear or branched. It stabilizes the protein, may increase the half-life and improve the bioactivity.
  • the agonist is a natural ligand of PAP or PAPlB or their receptor, or an antibody as defined above or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), that selectively binds PAP or PAPlB.
  • a further object of this invention is an antibody, or a fragment or derivative thereof, that selectively binds a PAP or PAPlB polypeptide or variant as disclosed above.
  • the antibody is a modulating antibody having the effect of changing the biological activity of PAP or PAPlB. Such modulating antibodies may be particularly valuable in the treatment or prevention of MS or related diseases.
  • the antibody, fragment or derivative thereof selectively binds PAP polypeptide having the sequence as defined by SEQ ID NO. 4 or SEQ ID NO. 5 or PAPlB polypeptide having the sequence as defined by SEQ ID NO. 8 or SEQ ID NO. 9 or SEQ ID NO. 11 or SEQ ID NO. 12.
  • the modulator is an inhibitor or antagonist of a PAP or PAPlB polypeptide.
  • a further object of this invention resides in a pharmaceutical composition
  • a pharmaceutical composition comprising a functional PAP or PAPlB polypeptide or a nucleic acid encoding a PAP or PAPlB polypeptide or a vector encoding the same, and a pharmaceutically acceptable diluent or carrier.
  • a further object of this invention resides in a pharmaceutical composition
  • a pharmaceutical composition comprising a modulator of PAP or PAPlB as defined above, and a pharmaceutically acceptable diluent or carrier.
  • the above uses or compositions are particularly suited for treating or preventing multiple sclerosis or a related disorder in a subject presenting an alteration in the PAP or PAPlB gene or polypeptide, particularly in a subject presenting a SNP as described in Table 2a, or 2b.
  • a functional PAP or PAPlB polypeptide or a nucleic acid encoding a PAP or PAPlB polypeptide or a vector encoding the same or a PAP or PAPlB modulator is intimately admixed with a pharmaceutical diluent or carrier according to conventional pharmaceutical compounding techniques known to the skilled practitioner.
  • the PAP or PAPlB modulator may be an antibody that selectively binds PAP or PAPlB or a natural modulator of PAP or PAPlB.
  • compositions according to the present invention can, for example, be administered orally or parenterally, i.e. intravenously, intramuscularly or subcutaneously, by inhalation or intranasally.
  • compositions suitable for oral administration can be solids or liquids and can, for example, be in the form of tablets, pills, dragees, gelatin capsules, solutions, syrups, chewing-gums and the like.
  • active ingredient may be mixed with an inert diluent or a non-toxic pharmaceutically acceptable carrier.
  • compositions which can release the active substance in a controlled manner.
  • Pharmaceutical compositions which can be used for parenteral administration, are in conventional form such as aqueous or oily solutions or suspensions generally contained in ampoules, disposable syringes, glass or plastic vials or infusion containers.
  • these solutions or suspensions can optionally also contain a sterile diluent such as water for injection, a physiological saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents, and agents for adjusting the osmolality.
  • a sterile diluent such as water for injection, a physiological saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents, and agents for adjusting the osmolality.
  • the amount of active ingredient in the pharmaceutical compositions can fall within a wide range of concentrations and depends on a variety of factors such as the patient's sex, age, weight and medical condition, as well as on the method of administration.
  • the daily dose can also fall within a wide range of dosage units. It should be understood that the specific doses can be adapted to particular cases depending on the individual requirements, at the physician's discretion.
  • Another object of this invention is an isolated or recombinant PAP gene or a fragment thereof, wherein said gene or fragment comprises a SNP selected from Table 2a or 2b.
  • the invention also relates to any vector comprising a nucleic acid as defined above.
  • the vector may be any plasmid, phage, virus, episome, artificial chromosome, and the like.
  • the vector is a recombinant virus.
  • Viral vectors may be produced from different types of viruses, including without limitation baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to recombinant DNA techniques known in the art.
  • the recombinant virus is typically replication-defective, even more preferably selected from El- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs.
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in
  • a further aspect of this invention is a recombinant host cell comprising a vector or a nucleic acid as defined above.
  • the recombinant cell may be any prokaryotic or eukaryotic cells as discussed above.
  • the recombinant cell preferably expresses a recombinant PAP or PAP 1 B polypeptide at its surface.
  • a preferred embodiment of the invention is the use of an activator or agonist of PAP or PAPlB in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder.
  • a particularly preferred embodiment of the invention is the use of an activator or agonist of PAP or PAPlB in the preparation of a medicament for the treatment of multiple sclerosis or a related disorder wherein the activator or agonist is an antibody as defined above or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • the invention also relates to a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a PAP or PAPlB gene or polypeptide as defined above.
  • a particular embodiment of the present invention resides in a method of treating or preventing multiple sclerosis or a related disorder in a subject, the method comprising (i) detecting in a sample from the subject the presence of an alteration in the PAP or PAPlB gene or polypeptide as defined above and (ii) administering to said subject an agonist of PAP or PAPlB.
  • said alteration is selected from the group consisting of an alteration as disclosed in Table 2a or 2b.
  • a method of treating multiple sclerosis, or a related disorder in a subject in need of such treatment comprising of administering to the subject a compound that modulates the synthesis, expression or activity of one or more of the genes or gene products of the genes listed in Table 2a or 2b in a therapeutically effective amount so that at least one symptom of the multiple sclerosis or a related disorder is ameliorated.
  • a method of treating multiple sclerosis or a related disorder in a subject in need of such treatment wherein the subject has a susceptibility alteration in a PAP or PAPlB gene comprising of administering to the subject a therapeutically effective amount of a medication for MS, such as for example interferon-beta, preferably interferon-beta Ia.
  • a medication for MS such as for example interferon-beta, preferably interferon-beta Ia.
  • the susceptibility alteration is selected from one or more of the susceptibility alterations listed in Table 2a or 2b.
  • the susceptibility alteration is a single nucleotide polymorphism.
  • MS samples The study comprised three collections of unrelated patients with MS, and unrelated healthy controls recruited from the neurological Department of Rennes (France: 314 cases; 353 controls), Huddinge (Sweden: 279 cases; 301 controls) hospitals and SeraCare (USA: 289 cases; 289 controls).
  • Table 1 provides a summary for the description and stratification study of the different collections.
  • RR relapsing-remitting
  • SP relapsing-secondary progressive
  • PP primary-progressive
  • RR relapses with full recovery or with a residual deficit and lack of progression between relapses
  • the female / male ratio in the patient group was 2.14 (214 Females & 100 Males) with a mean age of 44 [19;68] years and in the control group 2.07 (238 Females & 115 Males) with a mean age of 35 [18;56] years.
  • the female / male ratio in the patient group was 2.4 (196 Females & 83 Males) with a mean age of 47 [22;75].
  • the control group in Huddinge collection included 301 (214 Females & 87 Males) healthy volunteers and the Female / male ratio was 2.5. Ages ranged from 22 to 73 years with a mean age of 47 years.
  • the group of cases included 289 subjects with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 50 [32;74] years.
  • the group of healthy volunteers included 289 individuals with a sex ratio of 5.7 (246 females and 43 males) and a mean age of 48.7 [36;75] years.
  • Genomic DNA was extracted from EDTA anticoagulated peripheral blood according to a standard proteinase K digestion and a modified salting out extraction method of Miller and co-workers (1988).
  • Each group, or panel of 12 alterations must be of the same extension type for processing on the UHT since each extension mix contains two labeled terminators (Bodipy- Fluorescein and TAMRA). Each group of twelve is referred to as a panel of alterations.
  • 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.
  • a five-microliter PCR was performed in 384-well plates (MJ Research, Watertown, MA, USA) using 75-uM dNTPs and 0.5U AmpliTaq® Gold (Applied Biosystems) in IXPCR buffer. Two nanograms of genomic DNA were used in each reaction. The 24 PCR primers were pooled and added such that each was at a final concentration of 50 nM. Thermal cycling was performed in DNA Engine Tetrad thermal cyclers (MJ Research) using the following program: 95°C for 5 seconds followed by 45 cycles of 95°C for 30 seconds; 50°-55°C for 55 seconds; 72 0 C for 30 seconds.
  • the first six cycles used an annealing temperature of 50°C after which the annealing temperature was increased by 0.2°C in the subsequent cycles until the annealing temperature reached 55°C. After the last cycle, the reaction was held at 72°C for 7 minutes followed by a 4°C hold.
  • hybridization buffer 5M NaCl, 0.5 M EDTA, 580 mM morpholinoethane sulphonic acid (MES) pH 6.6, IX Denhardt's Solution
  • MES morpholinoethane sulphonic acid
  • the SNPstream Array Imager is based upon a two-laser, two-color approach. Each sample 5 is illuminated with a 488-nm laser beam and subsequently with a 532-nm laser beam to excite the fluorescent oligonucleotides captured on the UHT microarray plates.
  • the system contains two emission band filters. Fluorescence emission from 488-nm excitation (Bodipy- Fluorescein) is captured in a band 50 nm wide, centered at 535nm. Fluorescence emission from 532-nm excitation (TAMRA) is captured in a band 55 nm wide, centered at 10 590 nm.
  • a colorcorrected custom lens, of high numerical aperture and 100-um A 2 X3 well area is imaged per frame. Sixty- four 2 X3 well images/color are taken per plate for a total of 384 wells. Total time required for the process is approximately seven minutes/plate.
  • 15 Generation of genotype calls from spots detected using the SNPstream UHT Array Imager involves two discrete steps. First, the location and intensity of a spot within the well and plate is determined for each wavelength; second, a genotype call is made based on the relative fluorescent intensities of each spot. Once a genotype call has been made, results are written to an Oracle® database where the data can easily be retrieved for viewing.
  • 0 Spot detection is an automatic process performed by UHTImage software. Positive controls in each well are used to align the grids around the 4 X4 element array. Once a grid is drawn, each spot is analyzed for morphology (i.e., circular shape and regular pixel intensity across each spot). Spots with low intensity or unusual morphology are marked as empty or fail. For each spot that passes the morphology test, an intensity value is generated 5 and loaded into the UHT database. Failed spots are carried through the analysis but are flagged for the user to review.
  • morphology i.e., circular shape and regular pixel intensity across each spot.
  • Genotype calling is performed once all spot intensities are in the database for each sample within a plate.
  • Each SNP is analyzed separately using UHT GetGenossoftware. This software automatically creates genotype calls based on the intensity value of each spot at each wavelength for a given sample. These calls are based on how the sample points cluster when plotted on a X, Y graph where X corresponds to the intensity in the 488-nm channel and Y to that of the 532-nm channel. If a point falls between clusters or the intensity of the point is too low, the sample is failed. Otherwise the point is called as XX, XY, or YY with the X's and Y's being replaced by the actual allele calls (A,C,G,T).
  • UHT GetGenos uses a proprietary algorithm to determine the clusters and the genotypes for each sample. After the genotype calling, the results are stored in the database by microarray plate number, well, and spot location.
  • genomic DNA For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of Xbal or HmdIII (New England BioLabs) in volumes of 20 ⁇ L for 2 hours at 37 °C. Following heat inactivation at 70 °C for 20 minutes, 0.25 ⁇ M of Xbal adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA- 3') (Affymetrix), or HmdIII adaptor (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3' and 5 'phosphate -AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3') (Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25 ⁇ L for 2 hours at 16 °C.
  • T4 DNA Ligase
  • PCR contains 10 ⁇ L of the diluted ligation reactions (25 ng of starting DNA) in 100 ⁇ L volumes containing 1.0 ⁇ M of primer (5'-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3'), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfx Polymerase (Invitrogen), PCR Enhancer (Invitrogen) and Pfx Amplification Buffer (Invitrogen).
  • PCRs 30 cycles of PCRs are run with the following cycling program: 94 0 C denaturation for 15 seconds, 60 °C annealing for 30 seconds, and 68 °C extension for 60 seconds.
  • 3 ⁇ L of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons.
  • the PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 ⁇ L of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 ⁇ g per 45 ⁇ l.
  • the PCR products are fragmented to ⁇ 100 bp with DNAse I.
  • 0.20 U of DNAse I (Affymetrix) is added to 40 ug of purified PCR amplicons in a 55 ⁇ L volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels.
  • the 3' ends of the fragmented amplicons are biotinlyated by adding 214 ⁇ M of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 ⁇ L volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15 minutes.
  • Affymetrix a proprietary DNA labeling reagent
  • Affymetrix Terminal Deoxynucleotidyl Transferase
  • the fragmented and biotinylated PCR amplicons are combined with 11.5 ⁇ g/mL human Cot-1 (Invitrogen) and 1 15 ⁇ g/mL herring sperm (Promega) DNAs.
  • the DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt's solution, and 0.0115% Tween-20 in a final volume of 260 ⁇ L.
  • TMACl tetramethylamonium chloride
  • the hybridization solution was heated to 95 °C for 10 minutes then placed on ice.
  • Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X SSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 °C.
  • Hybridization signals are generated in a three step signal amplification process: lO ⁇ g/mL stfeptavidin 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 0 C.
  • SAPE stfeptavidin R-phycoerythrin
  • the SAPE and antibody were added to arrays in 6X SSPE, IX Denhardt's solution and 0.01% Tween-20 at 25 0 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
  • a stratification effect is a non-homogeneous representation of populations between the case and the control groups due to genetic heterogeneity, which may lead to spurious association results and replication problems.
  • cases and controls contain an admixture of different groups (for example, based on ethnicity), we expect to find a consistent pattern of allele-frequency differences between cases and controls, at many random loci throughout the genome, this difference exceeding the significant p-value for association at more than 5% of these random loci.
  • Fst test ( Wright 1951) is an ANOVA-based method. The Fst value quantifies the loss of heterozygosity due to existence of a hierarchical structure. If it is different from 0, it means that the population under study is genetically heterogeneous, since allelic frequencies are different between populations.
  • Pritchard & Rosenberg test (Am. J. Hum. Genet. 65:220-228, 1999) calculates an overall chi-square statistic of allelic frequency differences between cases and controls.
  • Genomic Control (Devlin and Roeder 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.
  • HWE Hardy-Weinberg law regulating equilibrium
  • control population used in case-control association studies must respect this equilibrium, if sampled randomly.
  • population of cases can present some disequilibrium that may point to "mutations" underlying the disease, since cases are not a random representation of the general population.
  • HWE test therefore serves two objectives: data review and quality check as well as detection of possible mutation.
  • Additional statistics include (i) the difference between allelic frequencies in cases and in controls (the larger the difference in allelic frequency for a given SNP, the more probable is an association between the genomic region containing that SNP and the disorder), (ii) the Odds Ratio (OR) of the association and (iii) the population Attributable Risk (pAR).
  • the "chosen” allele is the allele for which the frequency is increased in cases compared to controls.
  • Preferred single nucleotide polymorphisms indicative of multiple sclerosis are the chosen alleles of Tables 2a and 2b.
  • Mantel Haenszel test comparison of the significant findings across populations.
  • An OR higher than 1 shows that the probability of having multiple sclerosis respectively is higher when carrying the 'risk' allele [or genotype or haplotype] than when carrying the other ones.
  • EAE experimental autoimmune encephalitis
  • CFA Complete Freund's Adjuvant
  • Rats were euthanized and spinal cord samples were taken at different times during disease course: before immunogen injection (Gl), 6 days after immunization (G2), at the onset of the disease (G3), at the first peak of clinical symptoms (G4), at the first remission phase (G5) and the second remission phase (G7). After dissection spinal cord samples were frozen in liquid nitrogen. Number of animal per group is described in table 3.
  • RNA from each animal was extracted by RNeasy kit (Qiagen) followed by DNase I (Qiagen). After genomic control by qPCR on RNAs, 4 ⁇ g of each spinal cord total RNA were treated by 4units of RNAse free DNase I (Ambion). The cDNA was obtained using "Advantage RT for qPCR" kit (Clontech) following the instructions provided by the supplier.
  • PCR A twenty-mi croliter PCR was performed in 384-well plates (Applied Biosystems) using 25 ng cDNA, IX TaqMan universal PCR Master mix NO AmpErase UNG (Applied Biosystems), and IX primers Taqman(R) Gene Expression Assays (Applied Biosystems - 5 4331182) were used for PAP and REG3G.
  • HMBS hydroxymethylbilane synthase
  • IX Sybr Green PCR master mix Applied Biosystems
  • 300 nM mix primers forward and reverse were used.
  • primers details see Table 4. Experiments were performed in triplicate on 7900 Applied Biosystems machine. Thermal cycling was 0 performed using the following program: 95°C 10 minutes followed by 40 cycles of 95°C 15 secondes; 60°C 1 minute.
  • the Ct is an absolute value indicating the relative expression level of a gene.
  • a Ct under 20 is indicative of a highly expressed gene.
  • a Ct between 35 and 40 is indicative of a
  • - ⁇ Ct weakly expressed gene.
  • 2 allows to determine the expression of one gene between two tissues (one being the reference tissue, and the other 0 one the target tissue).
  • Gl control group
  • G2, G3, G4, G5, and G7 were normalized by HMBS housekeeping gene and by Gl .
  • the results of the quantitative expression analysis are shown in Table 5.
  • the PAP and 5 REG3G genes are expressed in rat spinal cord.
  • the expression level of PAP in the control group is weak (after 33.4 cycles of PCR).
  • PAP is significantly up-regulated in G4 in comparison with Gl (6.4 times) (see Table 5).
  • the expression level of PAPlB in the control group is very weak, close to the detection limit of the machine (after 38.4 cycles of PCR) and is strongly up-regulated in G2, G3, G4, G5 in comparison with Gl (up to 104.1 times in G3) (see Table 5).
  • Table 3 provides a summary for the description and distribution of the different groups.
  • Cytogenetic Band 4q33-q34 4q33-q34 8q22 Table 5 Expression of PAP, REG3G and HBMS genes in relapsing-remitting (RR) EAE rats spinal cord. Expression level expressed as a ratio obtained with normalization by HMBS and Gl (Group 1 : controls).
  • NA Non accurate values; only one sample in the group.
  • Example 3 PAP stimulates Myelin Basic Protein (MBP) production in maturating mixed spinal cord cultures
  • MBP myelin basic protein
  • the capture antibody mouse monoclonal antibody anti-MBP (1/5000; Chemicon), was diluted in PBS and incubated overnight at 4°C. The plates were blocked with PBS containing 1% BSA for 1 hour. Samples diluted in PBS were incubated for 2 hours.
  • the detection antibody rabbit polyclonal anti-MBP (1/300; Zymed) diluted in PBS-BSA, was incubated for 2 hours and revealed with an O-phenylenediamine Dihydrochloride (OPD) substrate after incubation with anti-rabbit horse-radish peroxidase-conjugated antibody (1/3000, Sigma; diluted in PBS-BSA, 1 hours).
  • OPD O-phenylenediamine Dihydrochloride
  • PAP increases MBP content, by increasing the number of oligodendrocytes, or by increasing their differentiation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

En général, la présente invention a pour objet des méthodes et des compositions pour détecter ou traiter des troubles inflammatoires, tels que la sclérose en plaques. Plus particulièrement, la présente invention concerne l'identification de gènes humains qui peuvent être utilisés pour diagnostiquer, prévenir et traiter une sclérose en plaques et des troubles apparentés ainsi que pour l'identification de médicaments thérapeutiquement actifs. L'invention concerne en outre des polymorphismes spécifiques du gène PAP qui sont apparenté à la sclérose en plaques, ainsi que des outils et des trousses de diagnostic basés sur ces polymorphismes. L'invention peut être utilisée pour le diagnostic d’une prédisposition à, une détection de, un traitement de et/ou la prévention de la sclérose en plaques et de troubles apparentés.
PCT/EP2006/012458 2005-12-22 2006-12-22 Compositions et méthodes pour traiter des troubles inflammatoires Ceased WO2007071437A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EPEP05112747.0 2005-12-22
EP05112747 2005-12-22
US75584206P 2006-01-03 2006-01-03
US60/755,842 2006-01-03

Publications (2)

Publication Number Publication Date
WO2007071437A2 true WO2007071437A2 (fr) 2007-06-28
WO2007071437A3 WO2007071437A3 (fr) 2007-09-20

Family

ID=37829289

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2006/012458 Ceased WO2007071437A2 (fr) 2005-12-22 2006-12-22 Compositions et méthodes pour traiter des troubles inflammatoires

Country Status (1)

Country Link
WO (1) WO2007071437A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2260857A1 (fr) 2009-06-11 2010-12-15 Alfact Innovation Nouvelles applications de HIP/PAP ou dérivés associés
US8012928B2 (en) 2008-12-19 2011-09-06 The Research Foundation Of State University Of New York Truncated PAP2 and methods of making and using same
US9388215B2 (en) 2013-03-15 2016-07-12 Shenzhen Hightide Biopharmaceutical, Ltd. Compositions and methods of using islet neogenesis peptides and analogs thereof

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935813A (en) * 1997-03-20 1999-08-10 Incyte Pharmaceuticals, Inc. Human pancreatitis-associated protein
FR2828693B1 (fr) * 2001-08-14 2004-06-18 Exonhit Therapeutics Sa Nouvelle cible moleculaire de la neurotoxicite
US20030170673A1 (en) * 2001-10-02 2003-09-11 Epstein Stephen E. Identification of genes involved in restenosis and in atherosclerosis
AU2003223526A1 (en) * 2002-04-24 2003-11-10 Georgtown University A gene abnormally expressed in autoimmune diseases and malignancies
JP2006503587A (ja) * 2002-05-16 2006-02-02 バンダービルト・ユニバーシティ 自己免疫疾患の予測方法
AUPS271902A0 (en) * 2002-05-31 2002-06-20 Griffith University Gene expression and multiple sclerosis
US20040156826A1 (en) * 2002-09-27 2004-08-12 Fernando Dangond Treatment of patients with multiple sclerosis based on gene expression changes in central nervous system tissues
EP1538218A1 (fr) * 2003-12-04 2005-06-08 Erasmus University Medical Center Rotterdam Méthode pour diagnostiquer ou examiner des maladies inflammatoires
JP2005160440A (ja) * 2003-12-05 2005-06-23 Hitachi Ltd 多発性硬化症に関連する遺伝子の発現測定方法、多発性硬化症関連遺伝子の発現を測定するためのチップ、多発性硬化症の罹患を判断するための遺伝子群、多発性硬化症の評価方法
US20050277593A1 (en) * 2004-05-24 2005-12-15 Washington University Therapeutic uses of Reg protein

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8012928B2 (en) 2008-12-19 2011-09-06 The Research Foundation Of State University Of New York Truncated PAP2 and methods of making and using same
EP2260857A1 (fr) 2009-06-11 2010-12-15 Alfact Innovation Nouvelles applications de HIP/PAP ou dérivés associés
WO2010142800A1 (fr) 2009-06-11 2010-12-16 Alfact Innovation Nouvelles applications de la protéine hip/pap ou de ses dérivés
US20120142604A1 (en) * 2009-06-11 2012-06-07 Alfact Innovation Novel applications of hip/pap or derivatives thereof
AU2010258565B2 (en) * 2009-06-11 2016-04-28 Alfact Innovation Novel applications of HIP/PAP or derivatives thereof
US9388215B2 (en) 2013-03-15 2016-07-12 Shenzhen Hightide Biopharmaceutical, Ltd. Compositions and methods of using islet neogenesis peptides and analogs thereof
US9738695B2 (en) 2013-03-15 2017-08-22 Shenzhen Hightide Biopharmaceutical, Ltd. Compositions and methods of using islet neogenesis peptides and analogs thereof
US10899815B2 (en) 2013-03-15 2021-01-26 Shenzhen Hightide Biopharmaceutical, Ltd. Compositions and methods of using islet neogenesis peptides and analogs thereof

Also Published As

Publication number Publication date
WO2007071437A3 (fr) 2007-09-20

Similar Documents

Publication Publication Date Title
AU2008318316B2 (en) RCA locus analysis to assess susceptibility to AMD and MPGNII
US20100120627A1 (en) Genemap of the human genes associated with psoriasis
US20100144538A1 (en) Genemap of the human genes associated with schizophrenia
CN106011243A (zh) 用于治疗、诊断和监测阿尔茨海默病的方法
Crowe et al. Candidate gene study of eight GABAA receptor subunits in panic disorder
CA2658563A1 (fr) Gene de susceptibilite a la maladie de crohn
Chagnon et al. Identification and characterization of an Xp22. 33; Yp11. 2 translocation causing a triplication of several genes of the pseudoautosomal region 1 in an XX male patient with severe systemic lupus erythematosus
US20080286876A1 (en) GENES ASSOCIATED WITH ALZHEIMER'S DISEASE - Hltdip
EP1448587B1 (fr) Gene du syndrome de noonan
US20100167285A1 (en) Methods and agents for evaluating inflammatory bowel disease, and targets for treatment
WO2007071437A2 (fr) Compositions et méthodes pour traiter des troubles inflammatoires
EP1863927A2 (fr) Compositions et methodes destinees a traiter et a diagnostiquer des troubles inflammatoires
US20140171371A1 (en) Compositions And Methods For The Diagnosis of Schizophrenia
US20120208718A1 (en) Schizophrenia treatment response biomarkers
EP1863926A2 (fr) Compositions et methodes destinees a traiter des troubles du snc inflammatoires
US20050255488A1 (en) NTRK1 genetic markers associated with age of onset of Alzheimer's Disease
US20080254451A1 (en) Compositions and Methods for Treating Schizophrenia and Related Disorders
EP1863937B1 (fr) Gene humain de susceptibilite a l'autisme codant une proteine transmembrane et ses utilisations
WO2009060066A1 (fr) Procédé de prédiction de la sensibilité thérapeutique de patients à un traitement médical par un interféron
CA2547033A1 (fr) Marqueurs genetiques de ntrk1 associes a l'evolution de la maladie d'alzheimer
US8236497B2 (en) Methods of diagnosing cardiovascular disease
EP1641937B1 (fr) Mutations dans le gene slc40a1 associees a l'homeostasie du fer alteree
KR20130027093A (ko) Ｋｌｏｔｈｏ 유전자의 단일염기다형을 이용한 심혈관계 질환 예측 방법
US20080194419A1 (en) Genetic Association of Polymorphisms in the Atf6-Alpha Gene with Insulin Resistance Phenotypes
JP4869834B2 (ja) 抗ヒトＴＮＦαキメラ抗体を含有する薬剤に対する副作用に関連する多型、およびその利用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06841129

Country of ref document: EP

Kind code of ref document: A2