WO2009037295A1 - Procédé pour tester une sensibilité au psoriasis - Google Patents

Procédé pour tester une sensibilité au psoriasis Download PDF

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WO2009037295A1
WO2009037295A1 PCT/EP2008/062401 EP2008062401W WO2009037295A1 WO 2009037295 A1 WO2009037295 A1 WO 2009037295A1 EP 2008062401 W EP2008062401 W EP 2008062401W WO 2009037295 A1 WO2009037295 A1 WO 2009037295A1
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psoriasis
allele
adam33
seq
nucleotide
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PCT/EP2008/062401
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Judith Fischer
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Commissariat A L'energie Atomique
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the invention relates to the field of psoriasis susceptibility, and preferably to a method of testing a subject thought to have or be predisposed to having psoriasis.
  • Psoriasis is a common hyperproliferative and chronic inflammatory skin disease with a prevalence of about 2-4% in Caucasian populations [NEVITT & HUTCHINSON, Br. J. Dermatol, vol.135, p:533-537, 1996].
  • Plaque psoriasis also known as psoriasis vulgaris, is by far the most common type of psoriasis, accounting for 80%-90% of all psoriasis patients. It appears as raised red scaling patches, most frequently on the elbows, knees, scalp and lower back.
  • This autoimmune disease is regarded as a multifactorial trait involving environmental factors such as intake of certain drugs, psychosocial stress, smoking, or climate conditions, all of which are well known triggering factors for primary manifestations or exacerbation in susceptible individuals [TAGAMI, Clin. Dermatol., vol.15, p:677-685, 1997].
  • TAGAMI Clin. Dermatol., vol.15, p:677-685, 1997.
  • twin and family studies which have shown a concordance rate of psoriasis in monozygotic twins of 65-72% vs 15-30% in dizygotic twins and a heritability of 80%
  • PSORSl Psoriasis Susceptibility 1
  • MIM 177900 contributes to the familial clustering of disease ( ⁇ ) to 33 ⁇ ⁇ ⁇ 50% [TREMBATH et al, abovementionned, 1997; The International Psoriasis Genetics Study, Am. J. Hum. Genet., vol.73, p:430-437, 2003]. Therefore, other susceptibility genes are likely to exist. Genome-wide linkage analyses have highlighted a number of disease loci on at least 15 chromosomes (see [LESUEUR et al, abovementionned, 2007] for review). Elucidation of the disease genes in these candidate loci is hampered by their large size and by the large number of candidates in each region.
  • the present invention relates to a method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for:
  • the present invention provides also a method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically inhibits the expression of ADAM33 gene to said subject.
  • the present invention provides also a method for treating and/or preventing psoriasis in a subject, comprising the administration of an effective amount of a compound which specifically increases the expression of ADAM33 gene to said subject.
  • the present invention provides also an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:
  • the present invention provides also an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:
  • Figurel shows the schematic representation of the psoriasis susceptibility locus on Chromosome 20pl3.
  • Table 1 shows SNPs genotyped at the ADAM33 locus and FBAT results for the univariate analysis. Table 2. Most significant under- and over-transmitted 3-SNP haplotypes for Region 2.
  • Table 3 shows the comparison of Set I and Set II
  • Table 4 shows the univariate SNP analysis in Set II and in combined sets. Only SNPs showing some association with psoriasis in Set I, in the univariate or in the 3-SNP haplotype analyses, were tested in Set II.
  • Supplementary Table Sl shows the results of the association tests for SNPs selected in Stage I.
  • Supplementary Table S2 shows the results for DEFB genes and for AK125948 gene.
  • Supplementary Table S3 shows the pairwise LD (D') for the 17 SNPs genotyped in the whole family set.
  • Supplementary Table S4 shows the results for HLA-Cw6 tagging SNPs (PSORSl locus).
  • Supplementary Table S5 shows the results for most significant under- and over- transmitted ADAM33 3-SNP haplotypes when stratifying according to HLA-Cw6 status in patients (for Set I)
  • the present invention is based on the discovery by the present inventors that particular single nucleotide polymorphisms (SNPs) of the ADAM33, and potentially ADAM33 alpha and/or beta iso forms expression, are correlated with psoriasis.
  • SNPs single nucleotide polymorphisms
  • the present invention provides a method of testing a subject thought to have or be predisposed to having psoriasis which comprises the step of analyzing a biological sample from said subject for :
  • Said method by detecting the presence of a SNP in the ADAM33 gene associated with psoriasis or by determining the expression of the ADAM33 gene in a subject enables to confirm that said subject has or is predisposed for having psoriasis.
  • the term "subject” refers to a mammal, preferably a human.
  • biological sample refers to solid tissues such as, for example, a lung biopsy; buccal swab, fluids and excretions such as for example, sputum, induced sputum, blood, serum, plasma, urine.
  • said biological sample is a fluid sample and most preferably a blood sample.
  • the expression "ADAM33 gene” refers to the ADAM metallopeptidase domain 33 which is well known from one of skill in the art.
  • the ADAM33 alpha isoform mRNAs has the sequence SEQ ID NO:1 (NM 025220) or the sequence SEQ ID NO:2 for ADAM33 beta isoform mRNA 2 (NM_153202)
  • the ADAM33 preproprotein alpha isoform has the sequence SEQ ID NO: 3 (NP 079496) or the ADAM33 preproprotein beta isoform has the sequence SEQ ID NO:4 for variant 2 (NP_694882)
  • the ADAM33 gene has the sequence SEQ ID NO:5 (NC 000020).
  • the expression of the ADAM33 gene refers to the expression of the alpha and/or beta of the ADAM33 gene.
  • ADAM33 gene associated with psoriasis As an example, one can cites rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis), rs628977 (nucleotide N at position 31 of SEQ ID NO:21, wherein allele T is associated to psoriasis), rs598418 (nucleotide N at position 27 of SEQ ID NO:22, wherein allele
  • said SNPs are selected in the group comprising or consisting of rs512625 (nucleotide N at position 31 of SEQ ID NO:6, wherein allele A is associated to psoriasis), rs677044 (nucleotide N at position 31 of SEQ ID NO:7, wherein allele G is associated to psoriasis), rs597980 (nucleotide N at position 31 of SEQ ID NO:8, wherein allele T is associated to psoriasis), rs44707 (nucleotide N at position 31 of SEQ ID NO:9, wherein allele C is associated to psoriasis)
  • ADAM33 may easily be equated or correlated with a similar mutation at a corresponding location for the ADAM33 gene from another species.
  • Typical techniques for detecting single nucleotide polymorphisms may include dynamic allele-specific hybridisation, ligation chain reaction, mini-sequencing, DNA"chips", allele-specific oligonucleotide hybridisation with single or dual-labelled probes merged with PCR or with molecular beacons, and others.
  • Analyzing the expression of the ADAM33 gene may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.
  • the expression of ADAM33 gene is assessed by analyzing the expression of ADAM33 alpha and/or beta mRNA transcripts or mRNA precursors, such as nascent RNA, of said gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a biological sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChipTM DNA Arrays (AFFYMETRIX).
  • the analysis of the expression level of mRNA transcribed from ADAM33 gene involves the process of nucleic acid amplification, e. g., by RT-PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol.87, p: 1874-1878, 1990), transcriptional amplification system (KWOH et al., 1989, Proc. Natl. Acad. Sci.
  • RT-PCR the experimental embodiment set forth in U. S. Patent No. 4,683, 202
  • BARANY Proc. Natl. Acad. Sci. USA, vol.88, p: 189-193, 1991
  • self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3 'regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • the expression of ADAM33 gene is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from ADAM33 gene.
  • an antibody e.g., a radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled antibody
  • an antibody derivative e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • ELISA enzyme linked immunoabsorbant assay
  • Polyclonal antibodies against ADAM 33 can be prepared by immunizing a suitable animal, such as mouse, rabbit or goat, with said protein.
  • a suitable animal such as mouse, rabbit or goat
  • the antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an ELISA using immobilized polypeptide.
  • antibody producing cells can be obtained from the animal and used to prepare monoclonal antibodies (mAb) by standard techniques, such as the hybridoma technique originally described by KOHLER and MILSTEIN (Nature, vol.256, p:495-497, 1975), the human B cell hybridoma technique (KOZBOR et al., Immunol, vol.4, p: 72, 1983), the EBV- hybridoma technique (COLE et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc, p: 77-96, 1985) or trioma techniques.
  • mAb monoclonal antibodies
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, COLIGAN et al. ed. , John Wiley & Sons, New York, 1994).
  • Hybridoma cells producing the desired monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA.
  • antibodies against ADAM33 can be already obtained from ABCAM, ABR- Affinity BIOREAGENTS, GENETEX, NOVUS BIOLOGICALS, or from SANTA CRUZ BIOTECHNOLOGY, INC.
  • the method of the invention may comprise comparing the level of expression of ADAM33 gene in a biological sample from a subject with the normal expression level of said gene in a control.
  • a significantly higher level of expression of said gene in the biological sample of a subject as compared to the normal expression level is an indication that the patient has or is predisposed to psoriasis.
  • a “higher level of expression” of ADAM33 gene refers to an expression level in a biological sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 20% superior to the normal level of expression of said gene, preferably at least 50% superior to the normal level of expression of said gene, and most preferably at least 100% superior to the normal level of expression of said gene.
  • a significantly lower level of expression of said gene in the biological sample of a subject as compared to the normal expression level is an indication that the patient has or is predisposed to psoriasis.
  • a “lower level of expression” of ADAM33 gene refers to an expression level in a biological sample that is lower than the standard error of the assay employed to assess expression, and is preferably at least 20% inferior to the normal level of expression of said gene, preferably at least 30% inferior to the normal level of expression of said gene, and most preferably at least 50% inferior to the normal level of expression of said gene.
  • the "normal" level of expression of ADAM33 gene is the level of expression of said gene in a biological sample of a subject not afflicted with psoriasis.
  • said normal level of expression is assessed in a control sample (e.g., sample from a healthy subject, which is not afflicted by psoriasis) and preferably, the average expression level of said gene in several control samples.
  • the present invention provides a use, for treating and/or preventing psoriasis in a subject, of a compound which specifically inhibits the expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms.
  • said compound specifically inhibiting the expression ADAM33 gene is an oligonucleotide, which is selected from the group comprising anti-sense RNA and DNA molecules, ribozymes, siRNAs and aptamers.
  • anti-sense RNA and DNA molecules and ribozymes function to inhibit the translation of ADAM33 mRNA.
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • anti-sense DNA oligodeoxyribonucleotides derived from the translation initiation site, e. g. , between - 10 and +10 regions of the ADAM33 nucleotide sequence, are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleo lytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of ADAM33 RNA sequences.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the anti- sense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • anti-sense cDNA constructs that synthesize anti-sense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • Short interference RNA molecules can also be used for inhibiting the expression of ADAM33 gene.
  • Said interference RNA molecules can be generated based on the genetic sequences of ADAM33 gene.
  • RNA interference is based on the degradation of particular target sequences by the design of short interference RNA oligo's (siRNA) which recognize the target sequence (here ADAM33) and subsequently trigger their degradation by a poorly understood pathway.
  • siRNA duplexes are shorter than 30 nucleotides, because longer stretches of dsRNA activate the PKR pathway in mammalian cells which results in a global a-specific shut-down of protein synthesis.
  • the length of said siRNA is comprised between 15 and 25 bp
  • siRNAs duplexes bases pair
  • siRNAs duplexes bases pair
  • RNA aptamers can also be used for inhibiting the expression of ADAM33 gene.
  • RNA aptamers have been used as therapeutic reagents for their ability to disrupt protein function. Selection of aptamers in vitro allows rapid isolation of extremely rare RNAs that have high specificity and affinity for specific proteins.
  • Exemplary RNA aptamers are described in U. S. Pat. No. 5,270,163, in GOLD et al. (Nature, vol.346, p:818-822, 1990), and TUERK & GOLD (Science, vol.249, p:505- 510, 1990).
  • RNA aptamers can bind to the three dimensional surfaces of a protein.
  • RNA aptamers can frequently discriminate finely among discrete functional sites of a protein (GOLD et al, Annu. Rev. Biochem., vol.64, p:763-797, 1995).
  • aptamers not only have the combined advantages of antibodies and small molecular mass drugs, but in vivo production of RNA aptamers also can be controlled genetically.
  • Such RNA expressing genes are usually smaller than protein- coding genes and can be inserted easily into gene therapy vectors.
  • said oligonucleotide is a siRNA.
  • the oligonucleotide may be delivered in vivo alone or in association with a vector.
  • a “vector” is any vehicle capable of facilitating the transfer of the siRNA to the cells.
  • such oligonucleotide can be associated with non-lipid cationic polymers (WU and WU, J. Biol. Chem., vol.263, p: 14621-4, 1988) or liposomes (BRIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989) to form complexes enhancing cellular uptake.
  • WU and WU J. Biol. Chem., vol.263, p: 14621-4, 1988
  • liposomes BTIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989
  • the present invention provides a use, for treating and/or preventing psoriasis in a subject, of a compound which specifically increase the expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta isoforms.
  • said compound specifically increasing the expression ADAM33 gene is an oligonucleotide expression vector coding for the ADAM33 protein, which vector may be for example in the form of a plasmid, a viral particle, a phage, etc.
  • Such vectors may include bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. Large numbers of suitable vectors are known to those of skill in the art and are commercially available.
  • Bacterial pQE70, pQE60, pQE-9 (QIAGEN), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHl ⁇ a, pNH18A, pNH46A (STRATAGENE), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (PHARMACIA).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (STRATAGENE), pSVK3, pBPV, pMSG, pSVL (PHARMACIA).
  • any other vector may be used as long as it is replicable and viable in the host.
  • the polynucleotide sequence preferably the DNA sequence in the expression vector coding for ADAM33 protein is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • prokaryotic or eukaryotic promoters such as
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription vector.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector of the invention containing the appropriate polynucleotide sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the polypeptide.
  • transcription of a DNA encoding for the polypeptide described previously by higher eukaryotes can be increased by inserting an enhancer sequence into the vector.
  • Enhancer are cis-acting elements of DNA, usually about from 10 to 300 pb that act on a promoter to increase its transcription. Examples of enhancer include the
  • SV40 enhancer the CMV early promoter enhancer, and adenovirus enhancers.
  • Such oligonucleotide expression vector can be associated with non-lipid cationic polymers (WU and WU, J. Biol. Chem., vol.263, p: 14621-4, 1988) or liposomes (BRIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989) to form complexes enhancing cellular uptake.
  • WU and WU J. Biol. Chem., vol.263, p: 14621-4, 1988
  • liposomes BTIGHMAN et al, Am. J. Med. ScL, vol.298, p: 278-81, 1989
  • an effective amount of compound is one which is sufficient to achieve a desired biological effect, in this case inducing an increased or decreased expression of ADAM33 gene, preferably from ADAM33 alpha and/or beta iso forms. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.
  • said compound specifically inhibiting or increasing the expression ADAM33 gene may be associated with a pharmaceutically acceptable vehicle.
  • the composition may comprise emulsions, microemulsions, oil-in-water emulsions, anhydrous lipids and oil- in-water emulsions, other types of emulsions.
  • the composition may also comprise one or more additives (e.g., diluents, excipients, stabilizers, preservatives). See, generally, Ullmann 's Encyclopedia of Industrial Chemistry, 6th Ed. (various editors, 1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et al, 1994, WILLIAMS & WILKINS).
  • the present invention provides an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:
  • ADAM33 gene in the cell contacted to that compound is ADAM33 gene in the cell contacted to that compound.
  • the present invention provides an in vitro method of selecting a compound, which can be useful for treating psoriasis, characterized in that said method comprises the steps of:
  • the term “compound” refers to any type of molecules such as polypeptides, polynucleotides, sugars, lipids, or any other chemical compounds.
  • ADAM33 Methods for determining the expression of the ADAM33 are well known from one of skill in the art. As an example, one can cite the methods which have been described previously. In the following, the invention is described in more detail with reference to nucleic acid sequences and the examples. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment, which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.
  • Set I corresponded to the 45 highly predisposed multigenerational families used for the initial genome-wide scan, and included on average 8 affected members per family [LESUEUR et al, abovementionned, 2007]
  • Set II corresponded to the 81 remaining smaller families (3 affected members per family on average, Table 3).
  • SNP selection SNPs were initially identified through the HapMap database. A list of 402 validated SNPs located between the microsatellite markers D20S864 and
  • D20S112 was generated.
  • biallelic markers were filtered according to the following criteria: selected markers had a minor allele frequency (MAF) > 20% in Caucasian populations, were located within known genes or nearby (+10 kb upstream and downstream of known genes), and SNPs with ambiguous flanking sequences were excluded for genotyping.
  • the population frequencies for the SNPs were taken from the CEU HapMap population (CEPH collection of Utah residents of northern and western European ancestry). Thus, 85 SNPs whose position was representative of the overall marker distribution were eligible for genotyping for Stage I. These SNPs were located in or near 65 different genes and were not in linkage disequilibrium with each other (1 SNP/ 137 kb on average).
  • SNP genotyping Genotyping was carried out using Taqman ® according to manufacturer's instructions. Primers and probes were supplied directly by Applied
  • Biosystems as Assays-by-DesignTM All assays were carried out in 384-well plates.
  • Each plate included negative controls (with no DNA) and positive controls were duplicated on a separate quality control plate. Plates were read on the ABI PRISM 7900 using the Sequence Detection Software (Applied Biosystems, Foster City, California, United States). Failed genotypes were not repeated.
  • Genotypes were checked for Mendelian inheritance errors using FBAT [LANG & LAIRD, abovementionned, 2002] and PEDSTATS was used to discard SNPs which deviate from Hardy- Weinberg Equilibrium in unrelated subjects [WIGGINTON & ABECASIS, Bioinformatics, vol.21, p:3445-3447, 2005].
  • the FBAT program to examine the transmission rates of marker alleles under the assumption of linkage.
  • the FBAT test is a multiallelic test based on the classic transmission/disequilibrium test (TDT) developed by SPIELMAN et al. [Am. J. Hum. Genet., vol.52, p:506-516, 1993]. It considers parents heterozygous for a certain allele at the marker locus associated with the disease and evaluates the frequency with which that allele is transmitted to affected offspring. In each trio, the untransmitted alleles of the parents serve as controls.
  • TTT transmission/disequilibrium test
  • HapMap data are available at "http://www.hapmap.org” (public data released n° 6 at 2005-03-01).
  • the dbSNP database is available at "http://www.ncbi.nlm.nih.gov/SNP/”.
  • PEDSTATS is available at "http://www.sph.umich.edu/csg/abecasis/PedStats/”.
  • the figure IA shows a genetic map on Chromosome 2Op 13 of a linkage interval associated with psoriasis (tel, telomere; cen, centromere). Position of microsatellites used in the linkage analysis is indicated in centimorgans (cM).
  • the 17Mb candidate locus on 2Op 13 extended from the telomere of the short arm of Chromosome 20 (D20S864) to the microsatellite D20S112 ( Figure IA), and contains 428 known genes. We aimed to define the boundaries of this region of linkage and to identify the causative variants. We initially selected 85 SNPs across the region for genotyping. For all SNPs, genotypes in founders satisfied the Hardy-Weinberg equilibrium.
  • Figure IB illustrates the results of the family-based association test (FBAT) under the assumption of linkage [LANG & LAIRD, Am. J. Hum. Genet., vol.71, p:575-584, 2002] for the 85 SNPs genotyped in 45 multigenerational families (corresponding to 295 nuclear families).
  • FBAT family-based association test
  • the figure IB shows the Z plot for association tests performed with FBAT under the assumption of linkage, for SNPs selected in Stage I. Position of SNPs is indicated in megabases (Mb). The dotted line indicates the threshold for significance of the association test (a Z score > 3 corresponds to a P ⁇ 0.05). Position, minor allele frequency, and FBAT results for all SNPs are given in Supplementary Table S 1.
  • the figure 1C. shows the detailed physical map on the contig NT Ol 1387.8 at the 3 candidate loci. Positions of the SNPs genotyped in stage I and in stage II are shown with arrows.
  • Red arrows indicate SNPs showing evidence for association (P ⁇ 0.05) in stage I; orange arrows indicate SNPs showing evidence for association (P ⁇ 0.05) in stage II; black arrows indicate SNPs showing no evidence for association in the univariate analysis.
  • HLA-Cw6 positive patients were identified using SNPs in strong LD with the risk allele (namely HCR-325 C>T (rsl30076), HCR-1723 G>T (rsl30079), HCR-2327 OG (rsl576) and CDSN971 OT (rsl062470) [24,25].
  • those markers define a strongly associated haplotype in our population (P ⁇ 0.000001 for haplotype H2, based on 1,000,000 permutations, Supplementary Table S4). Association between ADAM 33 and psoriasis was then monitored using FBAT when stratifying the families according to the presence or absence of this risk haplotype. Although the number of informative families was reduced, the associations between ADAM33 3-SNPs haplotypes and psoriasis were still observed in the group of patients not carrying HLA-Cw6, indicating that the 2 loci act independently. As expected, the associations were less significant in the group of patients carrying HLA-Cw6, due to the stronger contribution of the 6p21 locus to psoriasis susceptibility (Supplementary Table S5).
  • ADAM33 SNPs have been found to be associated with asthma and with bronchial hyper-responsiveness in Caucasians, in African Americans and in Hispanics [VAN EERDEWEGH et al, abovementionned, 2002; HOWARD et al, J. Allergy Clin. Immunol, vol.112, p:717-722, 2003 ; WERNER et al, Clin. Exp. Allergy, vol.34, p:26- 31, 2004].
  • An association of ADAM33 with allergic rhinitis has also been reported in the Japanese population [CHENG et al, Clin. Exp. Allergy, vol.34, p:l 192-1201, 2004].
  • This first report of an association between ADAM33 and psoriasis confirms that common biological pathways may be involved in the etiology of psoriasis and other clinically distinct immune-mediated diseases.
  • ADAM33 gene consists of
  • SNPs occur in the coding region of the gene and, of these, 3 are non- synonymous. We have excluded an association between two of them, T764M (rs2280091) and S774P
  • SNP 7 (rs677044) in the 3'UTR of ADAM33 showed some association when analyzed on its own and was present on all 4 most significant protective haplotypes (Table 2). However, this SNP was also present on other haplotypes not associated with the disease. Therefore, a functional role of this SNP in psoriasis susceptibility should be discarded. Finally, ADAM33 gene undergoes complex alternative splicing with several variant transcripts and their relative functional significance to each other is not clear [UMLAND et al, abovementioned, 2003, POWELL et al, Am. J. Respir. Cell. MoI Biol, vol.31, p:13-21, 2004].
  • ADAM33 polymorphisms may affect alternative splicing, splicing efficiency or mRNA turnover [VAN EERDEWEGH et al, abovementionned, 2002] but such functional effects for SNP5 (rs512625) in the 3' region O ⁇ ADAM33 and for SNP14 (rs597980) and SNP15 (rs44707) in intron 19 of the gene were not investigated in this study.
  • CAPNlO and NOD2 are two examples where haplotypes made up of non- coding variants have been associated with disease phenotypes in complex fashion while no association was seen in the univariate SNP analyses [COX et al, Diabetes, vol.53, p:S19-25, 2004]. The same situation is observed in the case of asthma and psoriasis, where the association with ADAM33 is stronger when combinations of SNPs are examined.
  • ADAM33 locus shows extended linkage disequilibrium upstream of ADAM33 to GFRA4, as well as downstream including SIGLECl (also named sialoadhesin SN) [WJST, Allergy, vol.62, p:444-446, 2007].
  • the region can be divided into 5 haplotype blocks, ADAM33 being situated between block 4 and 5, with an increased recombinatory rates around exons S to V of ADAM 33.
  • Half of the SNPs included in the associated combinations here lie in exon S or upstream (SNPs 5, 7, 9, 10, 11, 15, 16) and the second half lie downstream exon F (SNP 21, 23, 24, 25, 26, 27).
  • Psoriasis is a chronic disorder in which T-cell-mediated inflammation causes thickening of the skin.
  • T-cell-mediated inflammation causes thickening of the skin.
  • epidermal skin cells fail to mature into the flat, thickened, "cornified" layer they are supposed to. As a result, the epidermis tries to produce more cells than usual leading to the thickened epidermis, which then leads to inflammation.
  • ADAM proteins have a complex organization that includes a signal sequence and the following domains: pro, metalloprotease (including a zinc-binding sequence), disintegrin, cysteine-rich, epidermal growth factor, transmembrane, and cytoplasmic tail domains.
  • the proteins have diverse functions which include adhesion, cell fusion, intracellular signaling and the shedding of the extracellular portion of proteins such as cytokines and growth factors, leading to the soluble forms of these proteins.
  • Expression data suggest that ADAM33 is expressed in most human tissues, including skin [YOSHINAKA et ah, Gene, vo 1.282, p :227-236, 2002].
  • ADAM33 is relevant to the development of psoriasis because it may be involved in the inflammatory response, or in cell-cell and cell-matrix interactions that are essential for the development and maintenance of a tissue; likewise, extracellular matrix proteolysis is an important contributor to skin remodeling, which when altered might ultimately lead to significant desquamation or, perhaps, absence of cell maturation.

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Abstract

La présente invention porte sur un procédé de test d'un sujet dont on pense qu'il présente ou qu'il est prédisposé à présenter un psoriasis. Ce procédé comprend l'étape d'analyse d'un échantillon biologique provenant dudit sujet pour i) détecter la présence d'un SNP dans le gène ADAM33, lequel SNP est associé au psoriasis, et/ou ii) analyser l'expression du gène ADAM33.
PCT/EP2008/062401 2007-09-18 2008-09-18 Procédé pour tester une sensibilité au psoriasis WO2009037295A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032308A1 (fr) * 1994-05-20 1995-11-30 Board Of Regents, The University Of Texas System Compositions et leurs utilisations dans le diagnostic du psoriasis
US20020198371A1 (en) * 1999-08-09 2002-12-26 The Snp Consortium Identification and mapping of single nucleotide polymorphisms in the human genome
WO2006026222A2 (fr) * 2004-08-25 2006-03-09 Genentech, Inc. Disruptions geniques; compositions et methodes y relatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032308A1 (fr) * 1994-05-20 1995-11-30 Board Of Regents, The University Of Texas System Compositions et leurs utilisations dans le diagnostic du psoriasis
US20020198371A1 (en) * 1999-08-09 2002-12-26 The Snp Consortium Identification and mapping of single nucleotide polymorphisms in the human genome
WO2006026222A2 (fr) * 2004-08-25 2006-03-09 Genentech, Inc. Disruptions geniques; compositions et methodes y relatives

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 5 June 2004 (2004-06-05), "sqnm185783 Human DNA (Sequenom) Homo sapiens STS genomic, sequence tagged site.", retrieved from EBI accession no. EMBL:BV195764 Database accession no. BV195764 *
NELSON MATTHEW R ET AL: "Large-scale validation of single nucleotide polymorphisms in gene regions.", GENOME RESEARCH AUG 2004, vol. 14, no. 8, August 2004 (2004-08-01), pages 1664 - 1668, XP002506814, ISSN: 1088-9051 *
YAMADA R ET AL: "RECENT FINDINGS ON GENES ASSOCIATED WITH INFLAMMATORY DISEASE", MUTATION RESEARCH, AMSTERDAM, NL, vol. 573, 1 January 2005 (2005-01-01), pages 136 - 151, XP008048198, ISSN: 0027-5107 *

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