WO2014177699A1 - Inhibiteurs de rhoa (rock) pour le traitement d'une maladie intestinale inflammatoire - Google Patents

Inhibiteurs de rhoa (rock) pour le traitement d'une maladie intestinale inflammatoire Download PDF

Info

Publication number
WO2014177699A1
WO2014177699A1 PCT/EP2014/058997 EP2014058997W WO2014177699A1 WO 2014177699 A1 WO2014177699 A1 WO 2014177699A1 EP 2014058997 W EP2014058997 W EP 2014058997W WO 2014177699 A1 WO2014177699 A1 WO 2014177699A1
Authority
WO
WIPO (PCT)
Prior art keywords
ttc7a
deficiency
subject
cells
mutation
Prior art date
Application number
PCT/EP2014/058997
Other languages
English (en)
Inventor
Geneviève DE SAINT BASILE
Alain Fischer
Amélie BIGORGNE
Roxane LEMOINE
Hans Clevers
Henner FARIN
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Assistance Publique-Hôpitaux De Paris (Aphp)
Université Paris Descartes
Fondation Imagine
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 INSERM (Institut National de la Santé et de la Recherche Médicale), Assistance Publique-Hôpitaux De Paris (Aphp), Université Paris Descartes, Fondation Imagine filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2014177699A1 publication Critical patent/WO2014177699A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of inflammatory bowel diseases.
  • IBD Inflammatory bowel disease
  • Ulcerative colitis UC
  • Crohn disease CD
  • IBD 3 Ulcerative colitis
  • UC ulcerative colitis
  • CD Crohn disease
  • IBD interleukin-10
  • IL-10 interleukin-10
  • IL-10 receptor alpha and beta IL-10 receptor alpha and beta
  • XIAP is an apoptosis inhibitor that is involved in NOD2's downstream activation pathway 18 .
  • the present invention relates to a RhoA kinase (ROCK) inhibitor for use in the treatment of an inflammatory bowel disease in a subject in need thereof.
  • ROCK RhoA kinase
  • IBD inflammatory bowel disease
  • the inventors identified a homozygous hypomorphic missense mutation (c.211G>A leading to E71K) in the TTC7A gene in all affected family members.
  • the mutation was associated with impaired protein expression.
  • Partial TTC7A depletion modified proliferation, adhesion and migratory capacities in lymphocytes via inappropriate activation of the RhoA signalling pathway.
  • Growth and polarization of TTC7-deficient gut- epithelial-organoids was also found to be dependent on the RhoA signaling pathway.
  • TTC7A deficiency is a novel cause of IBD and is associated with combined immune deficiency in humans. It results from inappropriate activation of the RhoA signaling pathway, and thus inhibition of said pathway represents a novel therapeutic strategy for the treatment of IBD.
  • the present relates to a RhoA kinase (ROCK) inhibitor for use in the treatment of an inflammatory bowel disease in a subject in need thereof.
  • ROCK RhoA kinase
  • inflammatory bowel disease has its general meaning in the art and refers to any inflammatory disease that affects the bowel.
  • the term includes but is not limited to ulcerative colitis, and Crohn's disease.
  • Crohn's disease (CD)” or “ulcerative colitis (UC)” are chronic inflammatory bowel diseases of unknown etiology. Crohn's disease, unlike ulcerative colitis, can affect any part of the bowel.
  • the most prominent feature Crohn's disease is the granular, reddish-purple edmatous thickening of the bowel wall. With the development of inflammation, these granulomas often lose their circumscribed borders and integrate with the surrounding tissue. Diarrhea and obstruction of the bowel are the predominant clinical features.
  • IBD ulcerative colitis
  • IBD are characterized by abdominal pain, diarrhea (often bloody), a variable group of "'extra-intestinal'" manifestations (such as arthritis, uveitis, skin changes, etc.) and the accumulation of inflammatory cells within the small intestine and colon.
  • Additional symptoms, aspects, manifestations, or signs of IBD include malabsorption of food, altered bowel motility, infection, fever, rectal bleeding, weight loss, signs of malnutrition, perianal disease, abdominal mass, and growth failure, as well as intestinal complications such as stricture, fistulas, toxic megacolon, perforation, and cancer, and including endoscopic findings, such as friability, aphthous and linear ulcers, cobblestone appearance, pseudopolyps, and rectal involvement and, in addition, anti-yeast antibodies. See, e.g., Podolsky (2002) New Engl J. Med. 347:417-429; Hanauer (1996) New Engl. J. Med.
  • IBD affects both children and adults, and has a bimodal age distribution (one peak around 20, and a second around 40). IBD is a chronic, lifelong disease, and is often grouped with "autoimmune" disorders (e.g. rheumatoid arthritis, type I diabetes mellitus, multiple sclerosis, etc.). IBD is found almost exclusively in the industrialized world.
  • the subject has a TTC7A deficiency.
  • TTC7A has its general meaning in the art and refers to the tetratricopeptide repeat domain 7 A.
  • a human exemplary nucleic sequence is SEQ ID NO: 1.
  • a human exemplary amino acid sequence is SEQ ID NO:2.
  • 3301 cagcacagta cagacttctg gatctctctc aggtcttgcc cagggcggtc acaatgtgaa
  • ceamlilgkl hyvegsyrda ismyaragid dmsmenkply qmrllseafv ikglslerlp
  • TTC7A deficiency denotes that the cells of the subject or a part thereof have a TTC7A dysfunction, a low or a null expression of tetratricopeptide repeat domain 7A protein.
  • Said deficiency may typically result from a mutation in so that the pre-AR m is degraded through the NMD (non sense mediated decay) system.
  • Said deficiency may also typically result from a mutation (e.g. the mutation is 211G>A) so that the protein is misfolded and degraded through the proteasome.
  • Said deficiency may also result from a loss of function mutation leading to a dysfunction of the protein.
  • Said deficiency may also result from an epigenetic control of gene expression (e.g. methylation) so that the gene is less expressed in the cells of the subject. Said deficiency may also result from a repression of the TTC7A gene induce by a particular signalling pathway.
  • epigenetic control of gene expression e.g. methylation
  • the method of treatment of the present invention comprises a first step for determining whether the subject has a TTC7A deficiency.
  • the first step consists in detecting the mutation that is responsible for the TTC7A deficiency.
  • the presence of the mutation is 211G>A mutation may be looked for.
  • One skilled in the art can easily identify a mutation in TTC7A gene.
  • the mutation may be detected by analyzing a TTC7A nucleic acid molecule.
  • TTC7A nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance.
  • the nucleic acid sample may be obtained from any cell source or tissue biopsy.
  • Non-limiting examples of cell sources available include without limitation blood cells, buccal cells, epithelial cells, fibroblasts, or any cells present in a tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, such as blood, plasma, serum, lymph, etc.
  • DNA may be extracted using any methods known in the art, such as described in Sambrook et al, 1989.
  • R A may also be isolated, for instance from tissue biopsy, using standard methods well known to the one skilled in the art such as guanidium thiocyanate-phenol-chloroform extraction.
  • TTC7A mutations may be detected in a RNA or DNA sample, preferably after amplification.
  • the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a mutated site or that enable amplification of a region containing the mutated site.
  • reverse transcription and amplification by polymerase chain reaction (RT-PCR)
  • RT-PCR polymerase chain reaction
  • conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a particular TTC7A mutation.
  • RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • a cDNA obtained from RNA may be cloned and sequenced to identify a mutation in TTC7A sequence.
  • numerous strategies for genotype analysis are available (Antonarakis et al, 1989 ; Cooper et al, 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site.
  • a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation.
  • Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997).
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific oligonucleotides
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • InvaderTMassay or oligonucleotide ligation assay (OLA).
  • OLA may be used for revealing base substitution mutations.
  • oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation.
  • DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions where the mutations are located.
  • Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.
  • the mutation may be also detected at a protein level (e.g. for loss of function mutation) according to any appropriate method known in the art.
  • a biological sample such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of a mutated form of TTC7A protein, i.e. antibodies that are capable of distinguishing between a mutated form of TTC7A and the wild-type protein, to determine the presence or absence of a TTC7A specified by the antibody.
  • the antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies. Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred for they are more reproducible in the long run. Procedures for raising "polyclonal antibodies" are also well known. Alternatively, binding agents other than antibodies may be used for the purpose of the invention.
  • aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library.
  • the first step consists in determining the expression level of TTC7A gene in the biological sample obtained from the subject.
  • said biological sample is a blood sample or a PBMC sample or is a tissue sample resulting from a biopsy (e.g. an endoscopical biopsy performed in the colon of the subject).
  • the first step consist in i) determining the expression level of TTC7A gene, ii) comparing the level determined at i) with a predetermined reference value and iii) concluding that the subject has a TTC7A deficiency when the expression level determined at i) is lower than the predetermined reference value.
  • the predetermined reference value is the expression level determined in a healthy population of subject (e.g. the mean expression).
  • the expression level of a gene may be determined by determining the quantity of mR A.
  • Methods for determining the quantity of mR A are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a "detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315- 22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • SISH silver in situ hyhridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC-conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • probes labeled with fluorophores can be directly optically detected when performing FISH.
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200- 210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • Other methods for determining the expression level of a gene include the determination of the quantity of proteins encoded by said genes.
  • Such methods comprise contacting the sample with a binding partner capable of selectively interacting with a marker protein present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the binding partner may also be an aptamer.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • IHC immunohistochemistry
  • IHC specifically provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest.
  • a sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy.
  • Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling.
  • IHC systems include, for example, EnVision(TM) (DakoCytomation), Powervision(R) (Immunovision, Springdale, AZ), the NBA(TM) kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine(R) (Nichirei Corp, Tokyo, Japan).
  • a tissue section (e.g. a sample comprising cumulus cells) may be mounted on a slide or other support after incubation with antibodies directed against the proteins encoded by the genes of interest. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the proteins of interest.
  • IHC samples may include, for instance: (a) preparations comprising cumulus cells (b) fixed and embedded said cells and (c) detecting the proteins of interest in said cells samples.
  • an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffmation, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.
  • the TTC7A deficiency is detected in the subject by looking for a molecular or functional consequence of said deficiency.
  • said molecular consequence is an increased ROCK activity in the cells of the patients (e.g. the peripheral blood mononuclear cells (PBMC) or biopsy sample obtained from the subject).
  • PBMC peripheral blood mononuclear cells
  • the expression of the phosphorylated RhoA effectors molecule such as p-ERM and p-MLC may be determined such as described in the EXAMPLE, and compared to a predetermined reference value, wherein when the determined level is higher than the predetermined reference value it is concluded that the subject has an increased ROCK activity and thus has a TTC7A deficiency.
  • said functional consequences include but are not limited to a cytoskeletal polarization defect of the lymphocytes or of the intestinal epithelial cells, an increased proliferation and cell cycle progression of the lymphocytes or a growth impairment of gut organoids prepared from a biopsy sample obtained from the subject.
  • Said functional consequences may be detected as described in the EXAMPLE.
  • a biopsy sample e.g. an endoscopical biopsy from the colon of the subject
  • the polarization of the cells may be analysed by using antibodies or aptamers directed against proteins allowing the visualisation of basal, apical, or lateral membranes.
  • said proteins include but are not limited to actin, integrins, laminin, ZO-1 (zonula occludens) protein, villin, or alkaline phosphatase.
  • the formation of vacuoles in the cellular tissue may be detected by using the same antibodies or aptamers directed against actin, integrins, laminin, ZO-1 (zonula occludens) protein, villin, or alkaline phosphatise. Multilayered regions in the tissue may also be detected.
  • ROCK RasA kinase
  • ROCK is a member of the serine-threonine protein kinase family. ROCK exists in two iso forms, ROCK1 and ROCK2 (T. Ishizaki et al, EMBO J., 1996, 15, 1885-1893). ROCK has been identified as an effector molecule of RhoA, a small GTP-binding protein (G protein) that plays a key role in multiple cellular signaling pathways.
  • G protein GTP-binding protein
  • ROCK inhibitor refers to a natural or synthetic compound which inhibits ROCK1, and/or ROCK2 activity. In a particular embodiment the inhibitor is selective.
  • the selective ROCK inhibiting compounds are not limited to a particular manner of selective ROCK inhibition.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK1 activity over ROCK2 activity.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK2 activity over ROCK1 activity.
  • one or more of the selective ROCK inhibiting compounds selectively inhibit both ROCK1 activity and ROCK2 activity with similar capability.
  • ROCK inhibitors are well known in the art. For example, isoquinoline derivatives, especially fasudil, are typical ROCK inhibitors.
  • Fasudil (hexahydro-l-(5- isoquinolylsulfonyl)-lH-l,4-di-azepime), also named as HA-1077, is an isoquinoline sulfonamide derivative and the only clinically available ROCK inhibitor codeveloped by Asahi Kasei of Japan and Department of Pharmacology of Nagoya University. Hydroxyfasudil is an active metabolite of fasudil in vivo, which has higher affinity to ROCK than Fasudil.
  • Another isoquinoline derivative, H-1152P is optimized on the basis of fasudil.
  • Y-27632 Another type of ROCK inhibitor, inhibits both ROCK1 and ROCK2.
  • Optimization of these compounds leads to a more potent ROCK inhibitor, Y-39983, which is benefit for the treatment of the glaucoma (Kubo T, Yamaguchi A, Iwata N, The therapeutic effects of Rho-ROCK inhibitors on CNS disorders. Ther Clin Risk Manag 2008;4(3):605-15).
  • SLx-2119 a ROCK2-specific inhibitor, has recently been developed (Boerma M, Fu Q, Wang J, Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin.
  • ROCK inhibitors include those described in the international patent publications WO98/06433, WO00/09162, WO00/78351, WO01/17562, WO02/076976, EP1256574, WO02/100833, WO03/082808, WO2004/009555, WO2004/024717, WO2004/108724, WO2005/003101, WO20Q5/035501, WO2005/035503, WO2005/035506, WO2005/058891 , WO2005/074642, WO2005/074643, WO2005/Q80934, WO2005/082367, WO2005/082890, WO2005/097790, WO2005/100342, WO2005/103050, WO2005/105780, WO2005/108397, WO2006/044753, WO2006/051311, WO2006/057270, WO2006/058120 , WO2006/072792WO2011107608A1, and
  • the ROCK inhibitor is an inhibitor of ROCK expression.
  • An "inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • Inhibitors of gene 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 the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein (e.g. ROCK), 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 the target protein 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 gene expression for use in the present invention.
  • Gene expression can be reduced by contacting the tumor, 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 gene 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 Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al.
  • Ribozymes can also function as inhibitors of gene 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 the targeted mRNA sequences are thereby useful within the scope of the present invention.
  • Specific 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 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA 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. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • 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.
  • 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 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 R A 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 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). Such genetically altered 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 hematopoietic 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 operative ly 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 ROCK inhibitor of the invention is administered to the patient in a therapeutically effective amount.
  • a “therapeutically effective amount” of the ROCK inhibitor of the invention as above described is meant a sufficient amount of the compound. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the ROCK inhibitor 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” refers 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.
  • Galenic adaptations may be done for specific delivery in the small intestine or colon.
  • 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 ROCK inhibitor 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 antifusoluble 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 polypeptides in the required amount in the appropriate solvent with various 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.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. 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.
  • the ROCK inhibitor of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • 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.
  • FIGURES
  • Figure 1 ROCK inhibition normalizes apicobasal polarity in patient organoids.
  • A-E control- A, D
  • patient-derived ileum organoids B, C, E
  • Immunochemical staining of a6-integrin green
  • F- actin red
  • Nuclei were stained with DAPI.
  • Aberrant polarity is characterized by a multilayered epithelial structure and F-actin deposition around vacuoles (red arrows). Inverted polarity with F-actin deposition on the outer side and intra-epithelial a6-integrin staining (green arrows). All scale bars are 50 um.
  • EXAMPLE 1 SNP linkage analysis and sequence analysis.
  • Genomic DNA was isolated by phenol/chloroform extraction. RNA was isolated using an RNeasy Mini Kit (Qiagen). A genome-wide linkage study was performed using AffymetrixGeneChip Mapping 250 K Nspl, as described elsewhere ⁇ Homozygous regions were detected using a parametric, SNP -based linkage analysis (MERLIN software, version 1.1.1). Genomic DNA and cDNA were amplified, sequenced and analyzed on an ABI Prism 3700 system (using a BigDye Terminator sequencing kit from Applied Biosystems). Exome sequencing analysis of a DNA sample from an ELA patient was performed as described elsewhere 2 . Cell cultures.
  • Lymphoblasts were obtained by incubating PBMCs for 72 hours with phytohemagglutinin (PHA, 1 :400 dilution; Sigma- Aldrich) and IL-2 (50 IU/ml; PeproTech) in Panserin medium (Biotech GmbH) supplemented with 10% human AB serum (Etablatorium Francais du Sang). We then added more IL-2 (100 IU/ml) and cultured the cells for 6 to 7 days. The method for establishing organoid cultures from gut biopsies has been reported previously 3 .
  • the proportion of viable cells was determined by using the Annexin-V PE Apoptosis Detection Kit I (BD Biosciences). Cell fluorescence was measured on a FACSCanto (BD Biosciences).
  • Lymphoblasts primary fibroblasts and LBLs were lysed in radio- immunoprecipitation/glycerol buffer (50 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1% sodium deoxycholate) supplemented with protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Sigma-Aldrich).
  • Antibodies were purchased from eBioscience or BD Biosciences; standard flow cytometry methods were used for staining cell surface markers. Blood lymphocytes were stained with PeCy7-conjugated anti-CCR7, PE-conjugated anti-CXCR4, APC-conjugated anti-CD 11a, FITC-conjugated anti-CD 18 (all from BD Biosciences) and AL-57 antibody 4 . Data were collected on a FACSCanto system and analyzed with FlowJo8.8.4 software (TreeStar).
  • PBMCs from patients and controls were purified by density gradient centrifugation and cultured for three days with PHA (5 ⁇ g/mL) and for five days with tetanus toxoid antigen (0.125 lf/ml; Statens Serum Institute).
  • [ 3 H] thymidine was added for the last 18 hours.
  • Cell proliferation was determined as the cpm of [ 3 H] thymidine incorporated.
  • Proliferation was monitored by labeling T cells with 10 ⁇ CFSE (Invitrogen) prior to stimulation with anti- CD3/CD28 beads (Invitrogen), in accordance with the manufacturer's instructions.
  • Cell cycle analyses were performed by measuring the incorporation of the nucleoside analogue EdU into newly synthesized DNA two or five days after stimulation with anti- CD3/CD28 beads, PHA or PMA/ionomycin, in accordance with the manufacturer's instructions (Click- itEdU, Invitrogen). EdU incorporation was measured according to the abundance of fluorescent product and analyzed on a FACSCanto system with FlowJo 8.8.4 software. Lymphocyte-polarization assay.
  • Chemotaxis assays were performed using Transwell chambers (5 ⁇ pore size) with 200 ⁇ of cell suspension (1 x 10 6 cells/ml) in the top chamber and 600 ng/ml CCL21 or 1 ⁇ g/ml CXCL12 in the bottom chamber. After 3h at 37°C in 5% C0 2 , the proportion of migrated cells was calculated by flow cytometry. Cell adhesion and proliferation assays using xCELLigence technology (Roche). For the adhesion assay, the E-plate wells were coated with poly-L-lysine in the presence or absence of ICAM-1 or VCAM-1. 50 ⁇ of cell culture medium was added to the E-plate wells. The background signal was then measured.
  • PBMCs were resuspended in the appropriate cell culture medium, adjusted to 200,000 cells per 100 ⁇ and then incubated for 1 hour with a ROCK inhibitor (10 ⁇ Y27632). The cells were added to wells containing 50 ⁇ of medium. Adhesion of PBMCs was automatically monitored every 10 s over 4 hours.
  • For the proliferation assay 1500 fibroblasts from an ELA patient were incubated in the presence or absence of 10 ⁇ Y27632 for 1 hour and then added directly to E-plate wells containing 50 ⁇ of medium. After a 20 min incubation at room temperature, the E-plate was placed in the cell incubator. Lastly, cell proliferation was monitored every 10 min for up to 147 h. Electrical impedance was measured by the xCELLigence system's integrated software and expressed as a cell index. Rescue experiments.
  • TTC7A cDNA was subcloned into the EcoRl and BamHl restriction sites of the pEGFP-Cl plasmid (Clontech) using the following primers: TTC7A forward 5'-AGG AAT TCC ATG GCT GCG AAG GGC GCG CAC GGC-3'(SEQ ID NO:3) ; reverse 5'-GGG GAT CCC TCA GAG CTC TCT GGG GAT GAT GG -3' (SEQ ID NO: 4). Empty vector was used as a control. Primary fibroblasts (70% confluent) were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and Western blotting was performed 24h post-transfection.
  • Lipofectamine 2000 Invitrogen
  • Peripheral blood mononuclear cells were isolated from blood samples taken from enteropathy lymphocytopenia and alopecia (ELA) patients and controls.
  • B lymphoblast cell lines transformed with Epstein-Barr virus and fibroblast cell lines were established as described elsewhere 20 .
  • Cell proliferation, adhesion and migration and cell-cycle progression assays, Western blotting and the antibodies used to detect TTC7A, ROCK, phosphorylated ezrin/radixin/moesin (p-ERM), phosphorylated myosin light chain (p-MLC) and actin are described in EXAMPLE 1. Additional information on other assays, reagents and statistical techniques is also provided in EXAMPLE 1. Results
  • autoimmune hepatitis in B4 With age, four patients also developed autoimmune manifestations, autoimmune hepatitis in B4, autoimmune hemolytic anemia in A3, psoriasis and type I diabetes in B4 and autoimmune thyroiditis in CI .
  • the antrum was characterized by changes in the surface and foveolar epithelium that were exactly the same in biopsies from an 8 month-old patient (03) and a 50 year-old patient (CI).
  • the disorganized architecture of the epithelium consisted in tufting, loss of apical mucin, abnormal, pseudostratified cell organization and enlarged, hyperchromatic nuclei - all of which are characteristics of both degenerative and regenerative processes.
  • the lamina intestinal contained an inflammatory infiltrate with mononuclear cells and a high eosinophil count.
  • Antrum sections were Helicobacter py lori -negative. Colon lesions were severe and widespread. The epithelium was dedifferentiated and little mucus-secreting tissue remained. A combination of glandular necrosis, cell apoptosis and crypt abscesses in various sites was also noticed. The lamina intestinal was infiltrated by polymorphous inflammatory cells including mononuclear cells (CD4, CD8 T cells, B cells and macrophages) and polymorphonuclear (PMN) cells notably eosinophils.
  • CD4 T cells CD4, CD8 T cells, B cells and macrophages
  • PMN polymorphonuclear
  • TTC7A missense mutation did appear to have any influence on the steady- state level of mRNA TTC7A.
  • TTC7A was found to be ubiquitously present in a panel of cDNAs from a wide range of normal haematopoietic and non-haematopoietic tissues, with the highest levels in pancreas and thymus. Expression in the intestine was low but detectable. No significant difference in TTC7A expression levels was observed between T cell subsets.
  • TTC7A protein was not found in lymphoblastoid B cell lines (LBLs) nor in T lymphoblasts and was barely detectable in fibroblasts from ELA patients relative to control cells. These data indicate that the ELA syndrome was associated with the partial loss of TTC7A protein expression caused by a loss-of- function mutation in the TTC7A gene.
  • TTC7A belongs to a large family of tetratricopeptide repeat (TPR)-containing proteins.
  • the TPR domain is a 34 amino-acid consensus motif that is found in varying numbers of tandem repeats in different proteins 21 ' 22 .
  • Sequence analysis of TTC7A indicates that the E71 is highly conserved among TCC7 proteins and might be included in a degenerated TPR repeat.
  • the impaired cellular expression observed for the E71K mutant suggests that E71 is required for correct folding of TTC7A or an essential, stabilizing interaction with a partner.
  • TTC7A-deficient T cells Since polarization is required for cell migration, we also analyzed the migratory profile of TTC7A-deficient T cells. Impaired migration of TTC7A-deficient lymphocytes was observed in response to CCL21 and CXCL12, and was associated with a concomitant decrease in expression of the corresponding chemokine receptors CCR7 and CXCR4.
  • T cells' adhesion capacity of T cells was assessed. Adhesion to ICAM-1, and to a lesser extent VCAM-1, was consistently higher for the ELA patients' T cells than for controls' T cells. This difference in the patients' cells was associated with an increase of LFA-1 integrin expression. Expression of VLA-4 integrin (VCAM-1 ligand) was similar in cells from patients and controls. Expression of the high-affinity, open conformation of LFA-1 by activated or non activated patients' cells was not increased relative to controls (data not shown). Overall, these data suggest that the increased adhesion of TTC7A-deficient cells results from greater expression of B2-integrin and potentially higher affinity of VLA4 for VCAM1 , related to a modified cytoskeletal anchor. Increased proliferation and cell cycle progression of TTC7A-deficient cells
  • TTC7A-deficient patients had CD4 T cell lymphocytopenia associated with preserved T cell proliferation to mitogens but markedly impaired T cell proliferation to antigens. Further assessment of the proliferation potential of TTC7A deficient T cells was investigated in response to anti-CD3/CD28 stimulation, by using a sequential CFSE dilution assay.
  • the TTC7A-deficient T cells exhibited a significant increase in T cell proliferation between days 1 and 3. Accordingly, a high fraction of patient's T cells were in the S phase at day 2 of stimulation (as measured by EdU incorporation) and a corresponding lower proportion of cells in Gl phase, when compared with controls. Similar results were obtained when other stimuli were used (PHA or PMA/Ionomycin).
  • PBMCs from ELA patient T cells were incubated in the presence of the synthetic ROCK inhibitor Y27632.
  • the patients' PBMCs indeed showed expression of p-ERM and p-MLC at much the same level as control PBMCs.
  • Incubation of control PBMCs with Y27632 in the same setting did not have a significant effect on p-ERM or p-MLC expression.
  • Addition of Y27632 inhibited the hyperpro liferation of TTC7A-deficient T cells observed at day 3 after anti-CD3/CD28 stimulation.
  • a three-dimensional epithelial "organoid" culture 24 ' 25 was established from a rectum biopsy from an ELA patient (03).
  • the gut organoids displayed a disturbed epithelial architecture with condensed cell aggregates.
  • continuous addition of ROCK inhibitor (Y27632) resulted in lumen expansion and restored normal expansion of patient organoid.
  • the inhibitor was dispensable for the growth of control organoid 25 .
  • the flaky skin mice have a longer life spans and were thus analyzed in more details.
  • the flaky skin mouse phenotype overlaps (at least in part) with that of ELA patients.
  • the mice display pleiotropic skin abnormalities, with the development of thick white scales and patchy alopecia that turns into a papulosquamous disease resembling human psoriasis 28 ' 29 . Although only one adult ELA patients developed psoriasis, five displayed alopecia and four displayed onychopathy.
  • TTC7A lymphadenopathy
  • Both murine and human phenotypes display excessive T lymphocyte proliferation and an impaired cellular response to antigens.
  • Mouse vs. human disparities in phenotypic expression may be related to the time at which the assessment is carried out, the nature of the mutation, the disease's environment and, of course, species-specific differences.
  • the TTC7A protein does not have a known function. It contains 9 cognate TPR domains, which may be involved in multiple protein-protein interactions. Indeed, TPR domains are involved in a number of cellular processes, including cell-cycle
  • TPR-containing proteins disorders resulting from mutations in genes that encode TPR-containing proteins include Fanconi anemia 34 , Bardet-Biedl syndrome 35 , Charcot-Marie-Tooth disease type C 36 and tricohepatoenteric syndrome 37 . The latter is also characterized by the presence of a protracted gut disease. Nevertheless, relatively little is known about the function of proteins that contain TPR domains or the pathogenesis of conditions in which these proteins are defective. Here we show that TTC7A deficiency leads to inappropriate activation of the RhoA signaling pathway - a pathway that is known to orchestrate the interplay between the plasma membrane and the cortical cytoskeleton 38 .
  • TTC7A deficiency induces the phosphorylation of several ROCK effectors (such as ERM and the MLC) known to regulate cell adhesion and migration 23 ' 39 notably in hematopoietic cells 40 .
  • ROCK effectors such as ERM and the MLC
  • T lymphocytes from ELA patients adhered more strongly to integrin ligands and hence showed a lesser capacity to migrate in response to a range of chemoattractants.
  • Defective control of the RhoA signaling pathway activation also modulates the proliferation capacity of both lymphocytes and epithelial cells.
  • the mechanism by which TTC7A regulates the ROCK pathway remains to be established.
  • TTC7A deficiency a new cause of IBD that results from a loss of function mutation in TTC7A.
  • the mutation affects both lymphocyte and epithelial cell functions.
  • Our investigation of the in vivo and in vitro consequences of TTC7A deficiency show that this protein is critical for fine-tuning the cells' proliferation, adhesion and migration capacities and avoiding premature cell death.
  • TTC7A deficiency is associated with an increase in ROCK activity. The fact this increase can be reverted in vitro by treatment with a ROCK inhibitor suggests that this ELA syndrome is amenable to pharmacological manipulation.
  • Lymphocytes (per ⁇ X 10- J ) 2.9 3.9-9.0 1 .9 2.2 2.8 1 .9 1.4 3.4-9.0 2.8 2.3-5.4 0.7 0.5 0.80 1.4
  • CD4+ (%) 51 35-56 23 40 26 0.5 23 31 -56 21 6 28-47 36 25 49 31 -
  • CD4+CD45RA+CD31+ naive 33 60-72 35 63 71 47 60-72 47 50-85 11 1 1 2 43-
  • CD8+ (%) 25 12-23 33 30 0.8 19 2-24 33 3 16-30 57 35 25 18-
  • CD8+CD45RA-CCR7- 33 11 -20 2 10 11 -20 8 11 -20 2 65 85 20- re Treg CD4+CD25 hi FoxP3+ (%) 0.7 2-6 2.6 1.4 3.5-8 1.1 2.5-6 0.4 1.1 2.8- B T cell proliferation (cpm X 10- )
  • CD56+CD16+ (per ⁇ X 10- 3 ) 0.09 0.2-0.8 0.06 0.05 0.02 0.04 0.2-0.9 0.06 0.01 0.2-0.9 0.01 0.01 0.11 0.07-
  • CD19* ( ⁇ ⁇ ⁇ ⁇ - 3 ) 0.46 0.43-3.0 0.89 0.29 1 .1 0.5 0.7 0.6-2.6 1.0 0.05 0.7-2.6 0.04 0.09 0 0.09- CD27 + /CD19* memory (%) 3 7-27 2 1 .5 3 7-27 2 10-27 5 4 0 24-

Abstract

Dans la présente invention, les inventeurs ont maintenant identifié que la carence en TTC7A est une nouvelle cause de maladie intestinale inflammatoire. Elle résulte d'une activation inappropriée de la voie de signalement de RhoA, et ainsi, l'inhibition de ladite voie représente une nouvelle stratégie thérapeutique pour le traitement d'une maladie intestinale inflammatoire. Ainsi, la présente invention concerne un inhibiteur de kinase RhoA (ROCK) destinée à être utilisée dans le traitement d'une maladie intestinale inflammatoire chez un sujet qui en a besoin.
PCT/EP2014/058997 2013-05-03 2014-05-02 Inhibiteurs de rhoa (rock) pour le traitement d'une maladie intestinale inflammatoire WO2014177699A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP13305582 2013-05-03
EP13305582.2 2013-05-03

Publications (1)

Publication Number Publication Date
WO2014177699A1 true WO2014177699A1 (fr) 2014-11-06

Family

ID=48430633

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/058997 WO2014177699A1 (fr) 2013-05-03 2014-05-02 Inhibiteurs de rhoa (rock) pour le traitement d'une maladie intestinale inflammatoire

Country Status (1)

Country Link
WO (1) WO2014177699A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021191460A1 (fr) * 2020-03-27 2021-09-30 Universite De Paris Nouvelles cibles thérapeutiques à effet anti-inflammatoire et anti-interféron

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030134775A1 (en) * 1996-08-12 2003-07-17 Masayoshi Uehata Pharmaceutical agent containing Rho kinase inhibitor
WO2005074642A2 (fr) * 2004-01-30 2005-08-18 Smithkline Beecham Corporation Composes chimiques
WO2005082890A1 (fr) * 2004-02-20 2005-09-09 Smithkline Beecham Corporation Nouveaux composes
WO2005103050A2 (fr) * 2004-04-02 2005-11-03 Vertex Pharmaceuticals Incorporated Azaindoles utiles en tant qu'inhibiteurs de la proteine serine/threonine kinase superhelice de la famille rho (rock) et d'autres proteines kinases
EP1632492A1 (fr) * 2003-06-06 2006-03-08 Asahi Kasei Pharma Corporation Compose tricyclique
WO2008086047A1 (fr) * 2007-01-03 2008-07-17 Boehringer Ingelheim International Gmbh Inhibiteurs de la rho kinase
US20090325960A1 (en) * 2008-06-26 2009-12-31 Fulcher Emilee H Method for treating inflammatory diseases using rho kinase inhibitor compounds

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030134775A1 (en) * 1996-08-12 2003-07-17 Masayoshi Uehata Pharmaceutical agent containing Rho kinase inhibitor
EP1632492A1 (fr) * 2003-06-06 2006-03-08 Asahi Kasei Pharma Corporation Compose tricyclique
WO2005074642A2 (fr) * 2004-01-30 2005-08-18 Smithkline Beecham Corporation Composes chimiques
WO2005082890A1 (fr) * 2004-02-20 2005-09-09 Smithkline Beecham Corporation Nouveaux composes
WO2005103050A2 (fr) * 2004-04-02 2005-11-03 Vertex Pharmaceuticals Incorporated Azaindoles utiles en tant qu'inhibiteurs de la proteine serine/threonine kinase superhelice de la famille rho (rock) et d'autres proteines kinases
WO2008086047A1 (fr) * 2007-01-03 2008-07-17 Boehringer Ingelheim International Gmbh Inhibiteurs de la rho kinase
US20090325960A1 (en) * 2008-06-26 2009-12-31 Fulcher Emilee H Method for treating inflammatory diseases using rho kinase inhibitor compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SEGAIN JEAN-PIERRE ET AL: "Rho kinase blockade prevents inflammation via nuclear factor kappaB inhibition: Evidence in Crohn's disease and experimental colitis.", GASTROENTEROLOGY, vol. 124, no. 5, May 2003 (2003-05-01), pages 1180 - 1187, XP002699002, ISSN: 0016-5085 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021191460A1 (fr) * 2020-03-27 2021-09-30 Universite De Paris Nouvelles cibles thérapeutiques à effet anti-inflammatoire et anti-interféron
FR3108501A1 (fr) * 2020-03-27 2021-10-01 Universite De Paris « Nouvelles cibles thérapeutiques à effet anti-inflammatoire et anti-interféron ».

Similar Documents

Publication Publication Date Title
Anderson et al. Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence
Li et al. Deficient activity of the nuclease MRE11A induces T cell aging and promotes arthritogenic effector functions in patients with rheumatoid arthritis
Bruand et al. Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING
Lopez-Herrera et al. Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity
Guiducci et al. RNA recognition by human TLR8 can lead to autoimmune inflammation
Moens et al. Human adenosine deaminase 2 deficiency: A multi‐faceted inborn error of immunity
Snow et al. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations
US8114590B2 (en) Diagnosis and treatment of chronic lymphocytic leukemia (CLL)
Cobb et al. DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1
Cobb et al. Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations
Lemoine et al. Immune deficiency–related enteropathy-lymphocytopenia-alopecia syndrome results from tetratricopeptide repeat domain 7A deficiency
JP2018027959A (ja) 脱毛障害を処置するための方法
WO2011100841A1 (fr) Modulation de pd-1 et utilisations de celle-ci pour moduler la réplication du vih
Fiévet et al. Functional classification of ATM variants in ataxia‐telangiectasia patients
Raposo et al. Antiviral gene expression in psoriasis
KR20190095074A (ko) 뇌 종양 동물 모델 및 이의 제조 방법
WO2013155330A1 (fr) Compositions et procédés de caractérisation et de traitement d'une dystrophie musculaire
US20210186982A1 (en) Methods and compositions for treating melanoma
EP2695950A1 (fr) Marqueurs pour la réactivité à un inhibiteur du récepteur du facteur de croissance des fibroblastes
US20170016004A1 (en) DDX5 AND ASSOCIATED NON-CODING RNAs AND MODULATION OF TH17 EFFECTOR FUNCTION
US20190094229A1 (en) Methods of diagnosing, classifying and treating endometrial cancer and precancer
Wolf et al. UNC93B1 variants underlie TLR7-dependent autoimmunity
CN111187834B (zh) 胃肠道间质瘤的靶点depdc5及其在诊断和治疗中的应用
WO2014177699A1 (fr) Inhibiteurs de rhoa (rock) pour le traitement d'une maladie intestinale inflammatoire
Kim et al. Expression and secretion of CXCL12 are enhanced in autosomal dominant polycystic kidney disease

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: 14724045

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: 14724045

Country of ref document: EP

Kind code of ref document: A1