[go: up one dir, main page]

WO2014122199A1 - Méthodes et compositions pharmaceutiques pour traiter la pseudo-obstruction intestinale chronique - Google Patents

Méthodes et compositions pharmaceutiques pour traiter la pseudo-obstruction intestinale chronique Download PDF

Info

Publication number
WO2014122199A1
WO2014122199A1 PCT/EP2014/052296 EP2014052296W WO2014122199A1 WO 2014122199 A1 WO2014122199 A1 WO 2014122199A1 EP 2014052296 W EP2014052296 W EP 2014052296W WO 2014122199 A1 WO2014122199 A1 WO 2014122199A1
Authority
WO
WIPO (PCT)
Prior art keywords
rbpms2
expression
obstruction
subject
noggin
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/EP2014/052296
Other languages
English (en)
Inventor
Pascal De Santa Barbara
Jean-François GUICHOU
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.)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
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 Institut National de la Sante et de la Recherche Medicale INSERM, Universite de Montpellier filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2014122199A1 publication Critical patent/WO2014122199A1/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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • the present invention relates to methods and compositions for the treatment of chronic intestinal pseudo-obstruction.
  • Motility of the digestive tract is ensured by the contraction of visceral smooth muscles under the control of the autonomous enteric nervous system (ENS) and the interstitial cells of Cajal (ICC) (1-3). Dysfunction of just one of these cell types can lead to the development of gastrointestinal neuromuscular disorders in infants and adults (4, 5). Many studies have investigated the role of ENS damage in these diseases and shown that ENS absence caused by precocious differentiation or blockage of its migration triggers Hirschsprung disease (HSCR) (2-4). Alteration of ICC number, which perturbs the ICC network, also has been observed in different digestive motility disorders (4, 6). Until now, dysfunction of the downstream effector (visceral smooth muscle) rarely was investigated in gastrointestinal neuromuscular disorders (4, 6, and 7).
  • ENS autonomous enteric nervous system
  • ICC Cajal
  • Visceral SMCs originate from the splanchnopleural mesoderm that forms the primitive visceral mesenchyme via activation of the hedgehog/bone morphogenetic protein (BMP) pathway (2).
  • BMP hedgehog/bone morphogenetic protein
  • Differentiation of visceral mesenchymal cells into visceral SMCs can be visualized first through their elongation and clustering and later by the expression of SMC specific lineage markers, such as a-smooth muscle actin (aSMA), smooth muscle protein-22 (SM22), calponin, smoothelin, and smooth muscle myosin heavy chain (SMMHC), which precedes contractile function (8).
  • aSMA smooth muscle actin
  • SM22 smooth muscle protein-22
  • SMMHC smooth muscle myosin heavy chain
  • R As messenger R As
  • R A-protein complexes control multiple steps of this process, including mR A cellular localization, splicing, translational regulation, or mRNA degradation (12).
  • RBPs RNA-binding proteins
  • RRM RNA recognition motif
  • RNA-binding protein for multiple splicing 2 (RBPMS2), a member of the RRM family, is expressed in vertebrate heart and gastrointestinal tract (13, 15).
  • the present invention relates to a compound which is selected from the group consisting of RBPMS2 antagonists or RBPMS2 expression inhibitors for use in the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof.
  • the present invention also relates to a method of identifying a subject having a chronic intestinal pseudo-obstruction (CIPO) which comprises the step of analyzing a biological sample from said subject for determining the RBPMS2 expression level.
  • the inventors used avian replication-competent retroviral misexpression approaches to analyze the function of RBPMS2 in vivo and in primary cultures of chicken SMCs.
  • the inventors analyzed the expression of RBPMS2 transcripts in colon samples from pediatric patients with Hirschsprung's disease and patients with chronic pseudo obstruction syndrome (CIPO) with megacystis.
  • CIPO chronic pseudo obstruction syndrome
  • the present invention relates to a compound which is selected from the group consisting of RBPMS2 antagonists or RBPMS2 expression inhibitors for use in the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof.
  • CIPO chronic intestinal pseudo-obstruction
  • a subject denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with chronic intestinal pseudo-obstruction (CIPO) or digestive motility disorders, particularly those characterized by the presence of muscular lesions (visceral myopathies).
  • CIPO chronic intestinal pseudo-obstruction
  • digestive motility disorders particularly those characterized by the presence of muscular lesions (visceral myopathies).
  • the method of the invention may be performed for any type of chronic intestinal pseudo-obstruction (CIPO).
  • CIPO chronic intestinal pseudo-obstruction
  • the term "Chronic intestinal pseudo-obstruction” or “CIPO” as used herein refers to digestive motility disorders (visceral myopathies) that occurs in the gastrointestinal (GI or digestive) tract.
  • Chronic intestinal pseudo-obstruction (CIPO) refers to a rare and highly morbid syndrome characterized by impaired gastrointestinal propulsion together with symptoms and signs of bowel obstruction in the absence of any lesions occluding the gut lumen.
  • RBPMS2 has its general meaning in the art and refers to RNA-Binding Protein for Multiple Splicing-2 (SEQ ID NO: l) (Notamicola et al, 2012).
  • RBPMS2 RNA-Binding Protein for Multiple Splicing-2
  • RBPMS2 an early marker of SMC precursor cells and that ectopic expression of RBPMS2 in differentiated SMCs conducts to their dedifferentiation and triggers their proliferation.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., RBPMS2) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • proteins e.g., RBPMS2
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • RBPMS2 antagonist refers to a compound that selectively blocks or inactivates the RBPMS2.
  • RBPMS2 antagonist also refers to a compound that selectively blocks the binding of RBPMS2 to RNAs via its RRM domain.
  • RBPMS2 antagonist also refers to a compound that selectively blocks RBPMS2 binding to Noggin, inducing Noggin up-regulation and then inhibiting BMP signalling.
  • RBPMS2 antagonist also refers to a compound that is a RBPMS2 dimerization inhibitor.
  • the RBPMS2 antagonist of the invention is a RBPMS2 dimerization inhibitor.
  • the term “selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates RBPMS2 with a greater affinity and potency, respectively, than its interaction with the other sub-types or iso forms of the RBPMS family.
  • Compounds that prefer RBPMS2, but that may also block or inactivate other nuclear receptor sub-types, as partial or full antagonists, are contemplated.
  • a RBPMS2 antagonist is a small organic molecule, a peptide, a polypeptide, an aptamer or an intra- antibody.
  • the term “RBPMS2 dimerization inhibitor” refers to a compound that selectively prevents or blocks RBPMS2 dimerization.
  • RBPMS2 dimerization inhibitor refers to a compound that targets the residue Leucine 49 of RBPMS2 protein and blocks RBPMS2 dimerization.
  • RBPMS2 dimerization inhibitor also refers to a compound that targets the RRM-homodimeriztion motif (residues 47-50 of the SEQ ID NO: l).
  • a RBPMS2 dimerization inhibitor is a small organic molecule, a peptide, a polypeptide, an aptamer or an intra-antibody.
  • the RBPMS2 antagonist of the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against RBPMS2 of the invention as above described, the skilled man in the art can easily select those inhibiting RBPMS2 dimerization or inhibiting RBPMS2.
  • the compound of the invention is an inhibitor of RBPMS2 expression.
  • Inhibitors of RBPMS2 expression for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of RBPMS2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of RBPMS2 proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding RBPMS2 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Small inhibitory RNAs can also function as inhibitors of RBPMS2 expression for use in the present invention.
  • RBPMS2 gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that RBPMS2 expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • Ribozymes can also function as inhibitors of RBPMS2 expression for use in the present invention.
  • 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 endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RBPMS2 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of RBPMS2 expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis.
  • anti-sense R A molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing RBPMS2.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno- associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the compound of the invention may be administered sequentially or concomitantly with one or more muscular differentiation factors.
  • muscle differentiation factor refers to any muscular differentiation factor known to one of skill in the art to be effective to induce smooth muscle cell differentiation.
  • the muscular differentiation factors include but are not limited to bone morphogenetic protein such as BMP4, SRF and its co-factor Myocardin, a- smooth muscle actin (aSMA), smooth muscle protein-22 (SM22), calponin, smoothelin, and smooth muscle myosin heavy chain (SMMHC).
  • the present invention relates to a method of treating chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof, comprising the step of administering to said subject a compound which is selected from the group consisting of RBPMS2 antagonists or RBPMS2 expression inhibitors.
  • CIPO chronic intestinal pseudo-obstruction
  • the compound of the invention may be used or prepared in a pharmaceutical composition.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof.
  • CIPO chronic intestinal pseudo-obstruction
  • the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compound of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • parenteral administration such as intravenous or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
  • compositions of the invention may include one or more muscular differentiation factors.
  • said additional muscular differentiation factors may be contained in the same composition or administrated separately for simultaneous, separate or sequential use in the treatment of chronic intestinal pseudo-obstruction (CIPO). Screening method
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that are antagonists or inhibitors of expression of RBPMS2.
  • CIPO chronic intestinal pseudo-obstruction
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof, wherein the method comprises the steps of:
  • measuring the RBPMS2 activity involves measuring RBPMS2 dimerization level on the RBPMS2 cloned and transfected in a stable manner into a CHO cell line, human embryonic kidney (HEK) cell line or human CIPO cell line, measuring Noggin expression level, measuring the expression level of contractile proteins, determining the hypertrophic phenotype or measuring SMC contractility level in the presence or absence of the candidate compound. Tests and assays for screening and determining whether a candidate compound is a
  • RBPMS2 antagonist or RBPMS2 expression inhibitor are well known in the art. In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to reduce RBPMS2 activity. Activities of the candidate compounds, their ability to bind RBPMS2 and their ability to inhibit RBPMS2 activity may be tested using isolated smooth muscle cells (SMC) expressing RBPMS2, CHO cell line, human embryonic kidney cell line (HEK) or human CIPO cell line cloned and transfected in a stable manner by the human RBPMS2.
  • SMC smooth muscle cells
  • HEK human embryonic kidney cell line
  • CIPO cell line cloned and transfected in a stable manner by the human RBPMS2.
  • Cells and smooth muscle cells expressing another RNA-binding protein than RBPMS2 may be used to assess selectivity of the candidate compounds.
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of chronic intestinal pseudo-obstruction (CIPO) in a subject in need thereof, wherein the method comprises the steps of:
  • a candidate compound such as small organic molecule, intra- antibodies, peptide or polypeptide
  • step i) measuring the binding of the candidate compound to the polypeptide of step i) using appropriate biophysical techniques
  • the candidate compound binds to the amino acid residues 47-50 of the SEQ ID NO:l of the polypeptide and blocks polypeptides dimerization.
  • Methods for measuring the binding of the candidate agent to the polypeptide comprising amino acid residues 47-50 of the SEQ ID NO: l are well known in the art.
  • measuring the binding of the candidate agent to said polypeptide may be performed by biophysical techniques such as binding tests and crystallography.
  • a further aspect of the invention relates to a method of identifying a subject having a chronic intestinal pseudo-obstruction (CIPO) which comprises the step of analyzing a biological sample from said subject for:
  • detecting differential in the RBPMS2 expression level between the sample and the reference value is indicative of a subject having a chronic intestinal pseudo-obstruction (CIPO).
  • Analyzing the RBPMS2 expression level 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 RBPMS2 expression level is assessed by analyzing the expression of mR A transcript or mR A precursors, such as nascent R A, of RBPMS2 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 GeneChip(TM) DNA Arrays (AFFYMETRIX).
  • the analysis of the expression level of mRNA transcribed from the gene encoding for RBPMS2 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, 1991), self sustained sequence replication (Guatelli et al, 1990), transcriptional amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
  • RT- PCR the experimental embodiment set forth in U. S. Patent No. 4,683, 202
  • ligase chain reaction Barany, 1991
  • self sustained sequence replication (Guatelli et al, 1990)
  • transcriptional amplification system Kwoh e
  • 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 RBPMS2 expression level 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 the gene encoding for RBPMS2.
  • 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 reference value can be a threshold value or a cut-off value.
  • a threshold value can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • the person skilled in the art may compare the RBPMS2 expression levels (obtained according to the method of the invention) with a defined threshold value.
  • the threshold value is derived from the RBPMS2 expression level (or ratio, or score) determined in a biological sample derived from one or more subjects having a chronic intestinal pseudo-obstruction (CIPO).
  • CIPO chronic intestinal pseudo-obstruction
  • retrospective measurement of the RBPMS2 expression levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.
  • the reference value may consist in expression level measured in a biological sample associated with a healthy subject not afflicted with chronic intestinal pseudo-obstruction (CIPO) or in a biological sample associated with a subject afflicted with chronic intestinal pseudo-obstruction (CIPO).
  • CIPO chronic intestinal pseudo-obstruction
  • high RBPMS2 expression level is indicative of subject having a chronic intestinal pseudo-obstruction (CIPO) and low RBPMS2 expression level is indicative of subject not having a chronic intestinal pseudo-obstruction (CIPO).
  • CIPO chronic intestinal pseudo-obstruction
  • the present invention also relates to a method of treating chronic intestinal pseudoobstruction (CIPO) in a subject in need thereof comprising the steps of:
  • step iii) comparing said expression level measured in step ii) with a reference value, wherein high expression level of RBPMS2 is indicative of subject having a chronic intestinal pseudo-obstruction (CIPO), and
  • CIPO chronic intestinal pseudo-obstruction
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Sustained RBPMS2 expression alters chick gastrointestinal development, SMC differentiation, and contractile function.
  • FIG. 1 RBPMS2 overexpression hinders SMC differentiation and ability to contract.
  • FIG. 5 The impact of RBPMS2 misexpression on SMC differentiation in the presence or absence of BMP4.
  • Myc-tagged chick full-length RBPMS2 was cloned into the Replication-Competent Avian Leucosis Sarcoma virus strain A (RCAS [A]) vector to produce replication-competent retroviruses that express Myc-RBPMS2.
  • the RCAS(A)-Noggm and RCAS stain B with green fluorescence protein coding region (RCAS[B]-GFP) retroviral constructs were described previously (11,16). Retroviral constructs were transfected into the DF-1 chicken fibroblast cell line (ATCC-LGC) to produce retroviruses. Retroviruses were injected into the splanchnopleural mesoderm of stage- 10 chicken embryos to target the stomach mesenchyme (10,16). Eggs then were placed at 38°C until harvested.
  • avian retroviruses have a high tropism to infect SMCs and low tropism for enteric neurons.
  • SMC contractility was monitored with a Nikon inverted microscope and cells were imaged before and after treatment with 10 ⁇ 3 mol/L carbachol (Sigma, France), a muscarinergic agonist, as previously published (18).
  • carbachol Sigma, France
  • a muscarinergic agonist a muscarinergic agonist
  • E15 gizzards were cut at the level of the ventral and dorsal tendons to avoid damaging the muscular bundles (11).
  • Organs were mounted between 2 stainless steel hooks and placed in an organ bath filled with Tyrode- HEPES solution with 2.5 mmol/L CaCl 2 continuously bubbled with 95% 0 2 /5% C0 2 and maintained at 37°C. Changes in isometric tension were recorded using an IT 1-25 force transducer and an IOX computerized system (EMKA Technologies, Paris, France).
  • Gizzards initially were stretched at a resting tension of 0.5 g and, after a 60-minute equilibration period, contraction was induced with cumulative doses of carbachol (10 ⁇ 6 to 10 ⁇ 3 mol/L). Effects were evaluated by measuring the maximum tension, and data were expressed as changes relative to the basal tension (contraction in g) for 10 "4 mol/L carbachol concentration.
  • protein extracts (10 ⁇ g) were separated on 10% polyacrylamide gels (Bio-Rad Laboratories, France) and then blotted on nitrocellulose membranes.
  • Membranes were incubated with primary antibodies (anti-Myc from Ozyme [Montigny-Le-Bretonneux, France], anti-avian calponin from Sigma- Aldrich, anti- phosphoSMADl from Cell Signaling [Danvers, MA], anti-myocardin from Santa Cruz Biotechnology [Santa Cruz, CA], anti-glyceraldehyde-3 -phosphate dehydrogenase from Sigma- Aldrich, and anti-total AKT and anti-P-AKT from Cell Signaling) overnight and then with the relevant horseradish peroxidase-conjugated secondary antibodies. Detection was performed by chemiluminescence on Kodak films. Glyceraldehyde-3 -phosphate dehydrogenase expression was used to confirm equal loading.
  • RNAs were extracted from SMC primary cultures with the HighPure RNA Isolation kit (Roche Diagnostic, France) and reverse transcription was performed as described (11).
  • resulting complementary DNAs were biotinylated and hybridized to Affymetrix GeneChip Chicken Genome Arrays (Santa Clara, CA) following the manufacturer's protocols (IRB, CHRU Jardin, France) (11).
  • gene expression levels were measured using LightCycler technology (Roche Diagnostics). PCR primers were designed using the LightCycler Probe Design software 2.0. Each sample was assayed from 3 independent experiments performed in triplicate. Expression levels were determined with the LightCycler analysis software (version 3.5) relative to standard curves.
  • Data were represented as the mean level of gene expression relative to the expression of the reference gene ubiquitin. Data were analyzed using the Student t test and results were considered significant when the P value was less than .05 (*), P ⁇ .01 (**), or P ⁇ .001 (***).
  • RBPMS2 was expressed during chick gastrointestinal tract development.
  • RBPMS2 started to be expressed at embryonic day 4 (E4), an early stage of gastrointestinal tract development, in the regions of the future stomach, midgut, and colon.
  • E4 embryonic day 4
  • RBPMS2 was expressed strongly in the developing stomach, small intestine, and colon, with the exception of the cecum. Additional expression was observed in the developing lungs.
  • E6 RBPMS2 transcripts accumulated in the stomach, small intestine, and colon.
  • RBPMS2 expression was temporally and spatially comparable with that of aSMA, the earliest known SMC marker, which is expressed in the developing and differentiated visceral smooth musculature.
  • the inventors maintained RBPMS2 expression throughout visceral muscle development and differentiation by using an avian replication competent retroviral misexpression system that allows in vivo targeting of specific genes in the stomach mesenchyme (11, 16). Sustained RBPMS2 expression resulted in a dramatic alteration of the stomach morphology. Specifically, the proventriculus, which is the glandular part of the chick stomach, was hypertrophied, whereas the gizzard, the muscular part, was denser and malformed in comparison with controls that overexpressed GFP alone.
  • sustained RBPMS2 expression did not affect stomach development and patterning, as revealed by the normal expression of the mesenchymal marker BARXl and of the endodermal marker sonic hedgehog (SHH in RBPMS2 misexpressing stomachs.
  • SHH endodermal marker sonic hedgehog
  • the determination of visceral SMCs was not affected because positive aSMA cells still were observed in the smooth muscle layer; however, SMC differentiation was hindered as indicated by the reduction of calponin expression in RBPMS2 misexpressing stomachs in comparison with GFP controls.
  • HuC/D-positive neurons were organized into well-defined plexuses, suggesting that RBPMS2 misexpression did not induce detectable changes in ENS migration and differentiation.
  • RBPMS2 Inhibits BMP Signaling Through Induction of Noggin Expression
  • RBPMS2 the inventors analyzed the gene expression profiles of primary cultured SMCs infected with RCAS- RBPMS2 or RCAS-empty retroviruses for 3 days by microarray analysis.
  • RBPMS2 overexpression in differentiated SMCs induced the down-regulation of calponin and also of markers of SMC differentiation, such as caldesmon and SM-MHC.
  • Noggin the BMP signaling pathway inhibitor
  • BMP transcriptional targets including PITX2, ID2, and ID4 were down-regulated, suggesting a major inhibition of BMP activity.
  • Noggin up-regulation was confirmed by QPCR using primary SMCs harvested after 3 days of RCAS-RBPMS2 infection ( Figure 4).
  • the inventors performed in situ hybridization and showed that Noggin was expressed in gut mesenchymal derivatives as early as RBPMS2. Moreover, because misexpression of Noggin in stomach induces a hypertrophic phenotype (16) that is highly reminiscent of the defects observed upon RBPMS2 misexpression, the inventors monitored Noggin expression by in situ hybridization after RBPMS2 misexpression in the gastrointestinal mesenchyme as before. Noggin was strongly up-regulated in stomach and lung in comparison with controls. Conversely, misexpression of Noggin had a moderate impact on the spatiotemporal expression of RBPMS2. Altogether, these results show that RBPMS2 induces Noggin expression and accumulation in vivo and in primary cultured SMCs.
  • RBPMS2 can bind to R As via its RRM domain, the inventors investigated whether Noggin up-regulation in primary SMCs and in vivo upon RBPMS2 misexpression could be caused by interaction between RBPMS2 and Noggin.
  • Myc-tagged RBPMS2 from infected DF-1 cells was immunoprecipitated with anti-Myc antibodies bound to protein A Sepharose beads in the presence of total RNA from E6 gastrointestinal mesenchyme.
  • QPCR showed that Noggin was strongly amplified (6% of total Noggin mRNA) after immunoprecipitation of Myc-tagged RBPMS2, indicating that Noggin mRNA and RBPMS2 are present in a common RNA-protein complex.
  • PSMAD1 In control primary SMCs, high expression of PSMAD1 was associated with calponin expression.
  • calponin expression decreased concomitantly with a strong reduction of PSMAD1 expression, showing an inhibitory effect of RBPMS2 on the BMP signaling pathway, but also indirectly a positive effect of RBPMS2 on Noggin mRNA transcription or stabilization.
  • Noggin is an essential relay of RBPMS2
  • the inventors focused on Noggin function during visceral SMC development and differentiation.
  • the inventors thus misexpressed Noggin in stomach throughout visceral muscle development and analyzed the differentiation of visceral SMCs.
  • Noggin misexpression inhibited calponin expression in comparison with GFP controls, showing an alteration of visceral SMC differentiation without significant changes of the proliferative rate in vivo.
  • immunofluorescence analysis of primary cultured SMCs after 3 days of infection with RCAS-Noggin retroviruses showed that aSMA was expressed uniformly, whereas calponin expression was strongly decreased.
  • Noggin inhibits BMP signaling by interfering with homodimerization or heterodimerization of BMP ligands, thus blocking their interaction with receptors and preventing their activation (25). Because our results suggest that the RBPMS2 effect on calponin expression in SMCs is mediated through inhibition of the BMP pathway via Noggin induction, the inventors assessed the impact of RBPMS2 misexpression on SMC differentiation in the presence or absence of BMP4, the most strongly expressed BMP ligand in the gastrointestinal musculature (2,16,19). As previously observed in vascular SMC cultures, addition of BMP4 for 4 days increased differentiation of primary SMCs (19).
  • Intestinal motility disorders in infants comprise many heterogeneous diseases that are classified as gastrointestinal neuromuscular disorders and have clinical symptoms ranging from simple constipation to intestinal occlusion (4, 5). Recently, specific smooth muscle defects were shown to be involved in the pathogenesis of pediatric digestive motility disorders (7). Because our findings suggests that RBPMS2 might be involved in visceral SMC development and differentiation, the inventors analyzed the expression of RBPMS2 transcripts in colon biopsy specimens from pediatric patients with a history of chronic constipation associated with megacystis (CIPO), or aganglionosis (HSCR) and from neonates without digestive motility disorders (controls). Histologic analysis revealed the presence of regular circular and longitudinal smooth muscle layer in each case.
  • CIPO chronic constipation associated with megacystis
  • HSCR aganglionosis
  • RBPMS2 expression in the chick gastrointestinal mesenchymal layer is regulated temporally because it is high at E4-E6 and then progressively is reduced at a later stage.
  • This dynamic expression pattern corresponds to the progression of visceral undifferentiated mesenchymal cells into differentiated SMCs. Only few genes have such a dynamic expression pattern in visceral SMC precursors. SMA is expressed as early as RBPMS2, but then is maintained also in differentiating SMCs, when calponin, SM-MHC, and myocardin also are expressed. These data identify RBPMS2 as a marker of undifferentiated visceral SMCs.
  • RBPMS2 mainly in SMC primary cultures.
  • RBPMS2 but not Noggin, overexpression induced SMC proliferation and transient repression of aSMA expression. Therefore, in addition to the Noggin-BMP axis, RBPMS2 might regulate other pathways that contribute to the dedifferentiation and increased proliferation of visceral SMCs, thus dissociating the effect on proliferation from the effect on differentiation.
  • RBPMS2 and Noggin overexpression in differentiated SMCs hinders their differentiation associated with myocardin up-regulation. Indeed, recently, Yin et al (22) reported that myocardin accumulation inhibits its own function and that proteosomal degradation of myocardin is required for its full transcriptional activity. Similarly, the inventors show that myocardin accumulation results in a reduction of calponin expression (a marker of differentiated SMCs). In addition, RBPMS2 and Noggin misexpression led to up-regulation of the SRF target gene FHL2, which interacts with SRF and inhibits induction of smooth muscle contractile genes by SRF (21). Altogether, these results support the notion that activation of the RBPMS2/Noggin pathway inhibits SMC contractile genes through functional alteration of the myocardin/SRF pathway.
  • the inventors show that, in chick embryos, RBPMS2 is expressed during the early stages of visceral SMC development and that its expression is progressively lost during differentiation of visceral smooth muscles. Ectopic expression of RBPMS2 in primary culture of differentiated SMCs triggers an increase of their proliferative rate and hinders their contractile function, which also was observed at the organ level. The inventors show that regulated expression of RBPMS2 is important for the correct development and differentiation of visceral SMCs.
  • RBPMS2 transcript expression was significantly higher in circular smooth muscle cells from colon specimens of pediatric patients with CIPO (digestive dysmotility syndrome in the absence of physical obstruction of the bowel) (4-7, 26), whereas its expression was very low or absent in specimens from patients without digestive motility disorders or with HSCR (a developmental ENS disorder).
  • Some investigators have reported abnormal architecture of the tunica muscularis of colon specimens from patients with CIPO, suggesting a potential primary alteration of the visceral smooth muscles (6, 7).
  • the inventors identified RBPMS2 as a new marker of visceral SMC remodeling that could be useful for the characterization of smooth muscle alteration in visceral myopathies.
  • the human RBPMS2 cDNA sequence coding to the aminoacid 27 to 117 was subcloned into pET22 (pET22-human-RBPMS2-Nter). Substitution of Leucine by Glutamic Acid in position 49 of the human RBPMS2 sequence (L49E) was introduced by QuikChange site-directed mutagenesis method (Stratagene) in order to create pET22-human-RBPMS2- Nter L49E plasmid. The full-length human RBPMS2 cDNA was subcloned in the pCS2 vector with an in frame N-terminal HA tag and the CMV promoter (pCS2-HA-human- RBPMS2).
  • the full-length human RBPMS2 and RBPMS2 L49E were subcloned in the pHRTK vector with an in frame N-terminal Myc tag and the CMV promoter (respectively pHRTK-Myc-human-RBPMS2 and pHRTK-Myc-human-RBPMS2 L49E).
  • HA-tagged human TC10 was previously described (Coisy-Quivy et al, 2009).
  • Myc-tagged chick full- length RBPMS2 with corresponding Leu40Glu substitution was cloned into the RCAS vector to produce replication-competent retroviruses that express Myc-RBPMS2 Leu40Glu.
  • Myc- tagged chick full-length RBPMS2 (RCAS-Myc-gallus-RBPMS2), GFP (RCAS-GFP) and Myc-NICD were previously described (Notarnicola et al, 2012; Moniot et al, 2004; Shih and Holland, 2006). All plasmids were checked by DNA sequencing and protein expression.
  • DF1 cells transfected with different plasmid combination were labeled with mouse anti-HA (Santa Cruz Biotechnologies) and rabbit anti-Myc (Ozyme) primary antibodies and incubated with a pair of nucleotide-labeled secondary antibodies (rabbit PLA probe MINUS and mouse PLA probe PLUS; OLINK Biosciences, Uppsala Sweden) in hybridization solution.
  • secondary mouse and rabbit anti-IgG respectively coupled to Alexa 488 and 555 were incubated to detect protein expression.
  • NMR samples consist on approximately 0.5 mM 15 N - or 15 N, 13 C-labeled protein dissolved in 10 mM acetate buffer, 50 mM NaCl, pH 4.6 with 5% D20 for the lock. 1H chemical shifts were directly referenced to the methyl resonance of DSS, while 13 C and 15 N chemical shifts were referenced indirectly to the absolute 15 N /1H or 13 C/1H frequency ratios. All NMR spectra were processed and analysed with GIFA (Pons et al, 1996). Structures were validated using PROCHECK (Laskowski et al, 1993).
  • RBPMS2-Nter and RBPMS2-Nter L49E were labelled with the NHS ester of ATT0647N in 20 mM Na-phosphate buffer pH 7.5 with 50 mM KCl during 3 hours at room temperature. Labelled proteins were separated from the free dye using a 2 ml Zeba spin desalting column (Thermo Scientific) equilibrated in binding buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl). Anisotropy measurements were carried out at 25°C in dilution mode. RBPMS2-Nter-ATT03457N and RBPMS2-Nter L49E-ATT0647N (2nM final) were then mixed with different RNAs (2 ⁇ final) in binding buffer.
  • the mixture was serially diluted with the same buffer containing only 2nM RBPMS2-Nter-ATT0647N or RBPMS2-Nter L49E-ATT0647N. Measurements were made at each dilution in Corning black 384 wells assay plate with a TECAN Safire2 in polarization mode.
  • the avian DF-1 chicken fibroblast cell line (ATCC-LGC) was grown in DMEM supplemented with 10% FBS and transfected using Lipofectamine 2000 (Invitrogen, France) with above described constructs. Cells were analyzed after 24h. Cells were lysed using Lysis Buffer (20 mM Tris pH8, 50 mM NaCl, 1% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)).
  • the membrane was blocked with 10% nonfat milk in TBS + 0.1% Tween and probed with mouse anti-HA or rabbit anti-Myc polyclonal antibodies overnight. After several washes, membranes were incubated with the relevant horseradish peroxidase-conjugated secondary antibodies (Perkin Elmer). Detection was performed by chemiluminescence (Santa Cruz Biotechnologies) on Kodak films.
  • Retroviral constructs were transfected into the DF-1 chicken fibroblast cell line (ATCC-LGC) to produce retroviruses. Retroviruses were injected into the splanchnopleural mesoderm of Stage- 10 chicken embryos to target the stomach mesenchyme (Moniot et al, 2004; Notarnicola et al., 2012). Eggs were then placed at 38°C until harvested.
  • RCAS-empty or RCAS-Myc-gallus-RBPMS2, or RCAS-Myc-gallus-RBPMS2 L40E retroviruses and maintained in culture for 3 days.
  • avian retroviruses have a high tropism to infect SMCs.
  • anti-Myc Ozyme
  • anti-avian Calponin Sigma- Aldrich
  • anti-Phospho-Histone H3-Serl0 Millipore
  • Anti-digoxigenin antibodies coupled to alkaline phosphatase were used to detect Noggin sens/antisens complexes with BM Purple solution (Roche). Images were acquired using and a Carl-Zeiss Axiolmager microscope (for immunofluorescence) and a Nikon-AZlOO stereomicroscope (for whole- mount in situ hybridization).
  • the inventors looked at the structural organization of RBPMS2 by molecular modeling using the server @TOME-2 (Pons and Labesse, 2009). The inventors found that the N-terminus part of the human RBPMS2 protein (residues 27-117) was predicted to be structured as a RRM domain. Based on this result, the inventors have produced this domain in bacteria (RBPMS2-Nter) and purified it. Using NMR experiments, the inventors have confirmed the RRM fold for the N- terminus part of RBPMS2 protein, and found that this domain is exclusively present in the homodimeric form in solution.
  • the inventors conduct colmmunoprecipitation assays (colP) using DF1 cell lysates expressing Myc- or HA-tagged RBPMS2 proteins and anti-Myc antibodies. The inventors observe that HA-tagged RBPMS2 coprecipitates with Myc-tagged RBPMS2. To test whether the interaction is specific or mediated by bridging RNA, the inventors performed colP from RNase-treated assays and observe a specific homodimerization between RBPMS2 proteins. To confirm our results, the inventors include the small GTPase TCIO protein fused to HA tag as an additional negative control and observe no interaction with RBPMS2.
  • the inventors also investigate RBPMS2 homodimerization in cell culture using DuoLink technology, an in situ proximity ligation assay (PL A) that detects two proteins only when they are in close proximity.
  • the inventors find that HA-RBPMS2 interacts with Myc- RBPMS2 in DF1 cells expressing both Myc- and HA-RBPMS2 proteins.
  • PL A in situ proximity ligation assay
  • the inventors also test the interaction of RBPMS2 with unrelated Myc- or HA-tagged proteins, but we do not observe interaction. These data support that RBPMS2 is present as a homodimer in vivo.
  • RBPMS2 is a RNA-Binding Protein that can bind RNAs via its RRM domain and using immunoprecipitation of tagged avian RBPMS2 protein we found that Noggin mRNA and RBPMS2 are present in a common RNA-protein complex (Notamicola et al, 2012).
  • the inventors evaluate the capacity of human purified RBPMS2-Nter protein that contains the RRM domain to bind to the human NOGGIN mRNA synthesised in vitro by fluorescence anisotropy-based binding assays.
  • RBPMS2-Nter binds to human NOGGIN mRNA and identify that sequence between 518 to 838 is involved in this binding.
  • the inventors also find that human RBPMS-Nter L49E binds to similar NOGGIN sequence without affinity difference, suggesting that L49E substitution did neither alter its capacity to bind RNA nor its structure.
  • RBPMS2 or Myc-RBPMS2 or Myc-RBPMS2 L40E and anti-Myc antibodies The inventors observe that HA-tagged RBPMS2 coprecipitates with Myc-tagged RBPMS2 but faintly with Myc-tagged RBPMS2 L40E.
  • the Leucine to Glutamic Acid substitution abrogates 83,5% of the dimerization.
  • the inventors also test with DuoLink technique the impact of Leucine to Glutamic Acid substitution in cell culture. The inventors find that HA-RBPMS2 does not interact with Myc-RBPMS2 L40E in DF1 cells expressing both Myc- and HA-RBPMS2 proteins.
  • the inventors analyze the impact of RBPMS2 L40E in primary SMC cell culture and compare it to the action of RBPMS2.
  • the inventors establish primary cultures on Matrigel of visceral differentiated SMCs from E15 gizzard muscles in serum-free medium supplemented with insulin (Notamicola et al., 2012).
  • Control primary cultured SMCs in the presence of replication-competent retroviruses without transgene (RCAS-empty) were spindle-shaped and homogenously expressed aSMA and calponin, 2 SMC contractile markers, in highly organized filament bundles.
  • SMCs then were infected with replication-competent retroviruses (RCAS-RBPMS2 or RCAS-RBPMS2 L40E construct or RCAS-empty [control]) for 3 days. Although in control cells the expression of calponin remained unchanged, in SMCs infected with Myc-tagged RBPMS2 calponin expression is lost. The inventors also observe that SMCs infected with Myc-tagged RBPMS2 L40E calponin expression remain unchanged. The inventors also investigate the impact of RBPMS2 L40E on primary cultured SMCs with the analysis of the expression of phosphorylated Histone 3-SerlO (PH3), a standard marker of G2/M transition.
  • PH3 phosphorylated Histone 3-SerlO
  • the inventors analyze the impact of RBPMS2 L40E and RBPMS2 during the development of the avian gastrointestinal tract and compare it to the action of RBPMS2.
  • the inventors use the avian replication-competent retroviral misexpression system that allows in vivo targeting of specific genes in the stomach mesenchyme and the sustained expression of transgene throughout visceral muscle development and differentiation. As previously demonstrated (Notamicola et al, 2012), sustained RBPMS2 expression results in a dramatic alteration of the stomach morphology.
  • the proventriculus which is the glandular part of the chick stomach was hypertrophied, whereas the gizzard was denser and malformed in comparison with controls that overexpressed GFP alone.
  • the inventors previously showed that sustained RBPMS2 expression in the GI tract induces the upregulation of Noggin mRNAs in vivo (Notamicola et al, 2012).
  • the inventors analyze the impact of RBPMS2 L49E, RBPMS2 and GFP as control on Noggin mRNA expression by in situ hybridization.
  • the inventors observe that RBPMS2 misexpression in the gastrointestinal mesenchyme is always associated to the upregulation of Noggin mRNA in infected stomach in comparison to controls.
  • the inventors show that the conserved RBPMS2 protein, homodimerizes via its RRM domain and that this interaction is essential for its function.
  • the inventors also demonstrate that the newly identified RRM-homodimerization motif (residues 47-50 of the SEQ ID NO: 1) is crucial for the function of RBPMS2 at the cell and tissue levels.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des méthodes et des compositions pour traiter la pseudo-obstruction intestinale chronique.
PCT/EP2014/052296 2013-02-06 2014-02-06 Méthodes et compositions pharmaceutiques pour traiter la pseudo-obstruction intestinale chronique Ceased WO2014122199A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP13305144 2013-02-06
EP13305144.1 2013-02-06
US201361765825P 2013-02-18 2013-02-18
US61/765,825 2013-02-18

Publications (1)

Publication Number Publication Date
WO2014122199A1 true WO2014122199A1 (fr) 2014-08-14

Family

ID=47710075

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/052296 Ceased WO2014122199A1 (fr) 2013-02-06 2014-02-06 Méthodes et compositions pharmaceutiques pour traiter la pseudo-obstruction intestinale chronique

Country Status (1)

Country Link
WO (1) WO2014122199A1 (fr)

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
ANTONUCCI A; FRONZONI L; COGLIANDRO L ET AL.: "Chronic intestinal pseudo- obstruction", WORLD J GASTROENTEROL, vol. 14, 2008, pages 2953 - 2961
CÉCILE NOTARNICOLA ET AL: "The RNA-Binding Protein RBPMS2 Regulates Development of Gastrointestinal Smooth Muscle", GASTROENTEROLOGY, vol. 143, no. 3, September 2012 (2012-09-01), pages 687 - 697.e9, XP055054509, ISSN: 0016-5085, DOI: 10.1053/j.gastro.2012.05.047 *
DE SANTA BARBARA P; VAN DEN BRINK GR; ROBERTS DJ.: "Molecular etiology of gut malformations and diseases", AM J MED GENET, vol. 115, 2002, pages 221 - 230
DE SANTA BARBARA P; WILLIAMS J; GOLDSTEIN AM ET AL.: "Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development", DEV DYN, vol. 234, 2005, pages 312 - 322
FAURE S; DE SANTA BARBARA P; ROBERTS DJ ET AL.: "Endogenous patterns of BMP signaling during early chick development", DEV BIOL, vol. 244, 2002, pages 44 - 65
GABELLA G.: "Development of visceral smooth muscle", RESULTS PROBL CELL DIFFER, vol. 38, 2002, pages 1 - 37
GERBER WV; YATSKIEVYCH TA; ANTIN PB ET AL.: "The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart", MECH DEV, vol. 80, 1999, pages 77 - 86
GOLDSTEIN AM; BREWER KC; DOYLE AM ET AL.: "BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system", MECH DEV, vol. 122, 2005, pages 821 - 833, XP025329326, DOI: doi:10.1016/j.mod.2005.03.003
HAYASHI K; SAGA H; CHIMORI Y ET AL.: "Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase", J BIOL CHEM, vol. 273, 1998, pages 28860 - 28867
KNOWLES CH; DE GIORGIO R; KAPUR RP ET AL.: "Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group", ACTA NEUROPATHOL, vol. 118, 2009, pages 271 - 301, XP019713407
KNOWLES CH; DE GIORGIO R; KAPUR RP ET AL.: "The London Classification of gastrointestinal neuromuscular pathology: report on behalf of the Gastro 2009 International Working Group", GUT, vol. 59, 2010, pages 882 - 887
KRIEGLER: "A Laboratory Manual", 1990, W.H. FREEMAN C.O.
LAGNA G; KU MM; NGUYEN PH ET AL.: "Control of phenotypic plasticity of smooth muscle cells by bone morphogenetic protein signaling through the myocardin-related transcription factors", J BIOL CHEM, vol. 282, 2007, pages 37244 - 37255
LE GUEN L; NOTARNICOLA C; DE SANTA BARBARA P: "Intermuscular tendons are essential for the development of vertebrate stomach", DEVELOPMENT, vol. 136, 2009, pages 791 - 801
MAO J; KIM BM; RAJURKAR M ET AL.: "Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract", DEVELOPMENT, vol. 137, 2010, pages 1721 - 1729
MERICSKAY M; BLANC J; TRITSCH E ET AL.: "Inducible mouse model of chronic intestinal pseudo-obstruction by smooth muscle-specific inactivation of the SRF gene", GASTROENTEROLOGY, vol. 133, 2007, pages 1960 - 1970, XP022421704, DOI: doi:10.1053/j.gastro.2007.09.010
MONIOT B; BIAU S; FAURE S ET AL.: "SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals", DEVELOPMENT, vol. 131, 2004, pages 3795 - 3804
MURRY: "Methods in Molecular Biology", vol. 7, 1991, HUMANA PRESS, INC.
NAIR DG; HAN TY; LOURENSSEN ET AL.: "Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo", AM J PHYSIO GASTROINTESTINAL LIVER PHYSIO, vol. 300, 2011, pages 903 - 913
NOTARNICOLA C ET AL: "The RNA-binding protein RBPMS2 controls the development and the phenotypic plasticity of the visceral smooth muscle", NEUROGASTROENTEROLOGY AND MOTILITY, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 22, no. Suppl.1, August 2010 (2010-08-01), pages 90, XP009167974, ISSN: 1350-1925, [retrieved on 20100831] *
PHILIPPAR U; SCHRATT G; DIETERICH C ET AL.: "The SRF target gene Fhl2 antagonizes RhoA/MAL-dependent activation of SRF", MOL CELL, vol. 16, 2004, pages 867 - 880
PUIG I; CHAMPEVAL D; DE SANTA BARBARA P ET AL.: "Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction", J CLIN INVEST, vol. 119, 2009, pages 3586 - 3596
ROULEAU C; MATECKI S; KALFA N ET AL.: "Activation of MAP kinase (ERKI/2) in human neonatal colonic enteric nervous system", NEUROGASTROENTEROL MOTIL, vol. 21, 2009, pages 207 - 214
SANBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SANDERS KM; KOH SD; WARD SM.: "Interstitial cells of Cajal as pacemakers in the gastrointestinal tract", ANNU REV PHYSIOL, vol. 68, 2006, pages 307 - 343
ST JOHNSTON D: "Moving messages: the intracellular localization of mRNAs", NAT REV MOL CELL BIOL, vol. 6, 2005, pages 363 - 375
WALLACE AS; BURNS AJ: "Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract", CELL TISSUE RES, vol. 319, 2005, pages 367 - 382, XP019346457, DOI: doi:10.1007/s00441-004-1023-2
WEDEL T; VAN EYS GJ; WALTREGNY D ET AL.: "Novel smooth muscle markers reveal abnormalities of the intestinal musculature in severe colorectal motility disorders", NEUROGASTROENTEROL MOTIL, vol. 18, 2006, pages 526 - 538, XP003005589, DOI: doi:10.1111/j.1365-2982.2006.00781.x
WILMORE HP; MCCLIVE PJ; SMITH CA ET AL.: "Expression profile of the RNA-binding protein gene hermes during chicken embryonic development", DEV DYN, vol. 233, 2005, pages 1045 - 1051
XIN M; SMALL EM; SUTHERLAND LB ET AL.: "MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury", GENES DEV, vol. 23, 2009, pages 2166 - 2178, XP008116941, DOI: doi:10.1101/gad.1842409
YIN H; JIANG Y; LI H ET AL.: "Proteasomal degradation of myocardin is required for its transcriptional activity in vascular smooth muscle cells", J CELL PHYSIOL, vol. 226, 2011, pages 1897 - 1906

Similar Documents

Publication Publication Date Title
US9486521B2 (en) Therapeutic applications targeting SARM1
Notarnicola et al. The RNA-binding protein RBPMS2 regulates development of gastrointestinal smooth muscle
US20160025745A1 (en) Methods of diagnosing and treating fibrosis
US9347085B2 (en) Methods and compositions for reducing amyloid beta levels
Oh et al. Role of the PRC2-Six1-miR-25 signaling axis in heart failure
JP5737721B2 (ja) Ｈｔｒａ１変異と家族性虚血性脳小血管病との関連
Wang et al. Exosomes activate hippocampal microglia in atrial fibrillation through long-distance heart–brain communication
JP2011515072A (ja) α−シヌクレインキナーゼ
CA2584459A1 (fr) Modulation du facteur neurotrophique derive du cerveau (bdnf) issu de la nevroglie, dans le traitement et la prevention de la douleur
US12385921B2 (en) Methods for the diagnosis and treatment of gastrointestinal stromal tumors
US11219664B2 (en) Composition for diagnosing, preventing, or treating vascular smooth muscle cell proliferative diseases using FGF12
WO2014122199A1 (fr) Méthodes et compositions pharmaceutiques pour traiter la pseudo-obstruction intestinale chronique
US20230375528A1 (en) Screening method for the identification of novel therapeutic compounds
WO2020146639A2 (fr) Compositions et méthodes pour le diagnostic et le traitement de maladies du foie
US20150259403A1 (en) Methods and Pharmaceutical Compositions for Treatment of Gastrointestinal Stromal Tumors
US7402406B2 (en) Methods of evaluating phosphatase inhibitors
KR102279751B1 (ko) 근원성 인자 또는 nogo-a 측정 제제를 포함하는 근육병 진단용 조성물과 이를 이용한 근육병 진단 방법
Zhang et al. STAT3-mediated ferroptosis is involved in α-synuclein pathology
Fard Semaphorin 6C controls pancreatic cancer cell viability and growth
US20090047259A1 (en) Methods of Using the Calcineurin A Variant CnA-beta 1
WO2023120612A1 (fr) Agent thérapeutique ou prophylactique pour la crise cardiaque, la fibrose cardiaque ou l'insuffisance cardiaque, dans lequel htra3 est la cible thérapeutique
WO2022195042A1 (fr) Protéine ayant une fonction de hdgf (facteur de croissance dérivé de l'hépatome) pour une utilisation dans le traitement et la prévention de maladies neurodégénératives
Yan et al. Breaking the Chains of Vascular Stiffness: The Role of Fibulin-1 and its Regulatory Network
JP2015525770A (ja) 血管再構築のためのＮｋｘ２．５阻害剤
WO2008125816A1 (fr) Modulation de la prolifération cellulaire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14703342

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14703342

Country of ref document: EP

Kind code of ref document: A1