WO2008151289A1 - Modulation d'une inflammation des voies respiratoires - Google Patents

Modulation d'une inflammation des voies respiratoires Download PDF

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WO2008151289A1
WO2008151289A1 PCT/US2008/065951 US2008065951W WO2008151289A1 WO 2008151289 A1 WO2008151289 A1 WO 2008151289A1 US 2008065951 W US2008065951 W US 2008065951W WO 2008151289 A1 WO2008151289 A1 WO 2008151289A1
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activity
agent
dcplβ
pathway
cells
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Frank Mckeon
Hongmei Mou
Annie Yang
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors

Definitions

  • Airway inflammation is a significant mediator of respiratory disorders. Aspects of airway inflammation can be attributed to hyperactivation of innate immune signals, with or without pathogen induction. Hyperactivation of these signals has been implicated in the severity of asthma, cystic fibrosis, and COPD.
  • this disclosure features a method of inhibiting airway inflammation in a subject, e.g., a patient.
  • the method includes increasing the p73/DCPl ⁇ pathway activity in airway cells of the subject.
  • the p73/DCPl ⁇ pathway activity can be increased in a variety of ways, e.g., by one or more of: (i) reducing p73 degradation, (ii) increasing p73 levels or activity, and (iii) increasing DCP l ⁇ activity.
  • the method can include administering, to the subject, an agent that increases p73/DCPl ⁇ pathway activity.
  • the agent can be administered by an inhalatory route.
  • the method can include other features described herein.
  • the agent includes a nucleic acid encoding a protein with p73 activity or DCPl ⁇ activity.
  • the nucleic acid includes a cilia-specific promoter and a sequence encoding a protein with p73 activity or DCPl ⁇ activity.
  • the nucleic acid can be delivered using a gene therapy vector.
  • the agent includes a proteasome inhibitor, e.g., an inhibitor that reduces p73 degradation.
  • the methods and compositions described herein can also be used to reduce activation of an immune response (e.g., the innate immune response) in a subject, particularly in ciliated airway cells, and/or to decrease cytokine expression in a mammalian cell, e.g., a ciliated airway cell.
  • an immune response e.g., the innate immune response
  • the disclosure features a method of evaluating an agent. The method includes: contacting the agent to the cell; and evaluating activity of the p73/DCPl ⁇ pathway. An increase in pathway activating indicates that the agent can reduce inflammation or cytokine expression.
  • activity of the p73/DCPl ⁇ pathway is evaluated by evaluating DCPl ⁇ expression, e.g., by evaluating DCPl ⁇ mRNA or protein levels.
  • activity of the p73/DCPl ⁇ pathway can be evaluated by evaluating a reporter gene whose expression is operably linked to a transcriptional regulatory region of the DCP l ⁇ promoter.
  • the method can further include evaluating the agent in an animal, e.g., a non- human animal such as a transgenic non-human animal having modified p73 gene activity.
  • the animal can be an animal model of airway inflammation.
  • the method can further include one or more of: preparing the agent in a pharmaceutical composition and administering the agent to a patient.
  • agents identified by a method described herein and pharmaceutical compositions that include an agent identified by a method described herein.
  • the disclosure features a method that includes obtaining a sample that includes ciliated cells from a mammalian subject and evaluating the p73/DCPl ⁇ pathway or a component of the p73/DCPl ⁇ pathway in one or more cells in the sample.
  • the method can be used, e.g., to diagnose the extent of the inflammatory response in the subject.
  • the disclosure features a method that includes selecting an agent that modulates the p73/DCPl ⁇ pathway and administering the agent to a subject who has airway inflammation.
  • a related method includes selecting such an agent, identifying a modified form or variant of the agent, and preparing the modified or variant form as a pharmaceutical composition and/or administering the modified or variant form to a subject.
  • the disclosure features an isolated nucleic acid that includes a cilia-specific promoter and a sequence encoding a protein that increase p73/DCPl ⁇ pathway activity.
  • the promoter is operably linked to the coding sequence.
  • the coding sequence can encode a protein having p73 or Dcpl ⁇ activity.
  • a gene therapy vector that includes the nucleic acid can be prepared.
  • the gene therapy vector can be packaged in viral particles for delivery to a cell.
  • the p73/DCPl ⁇ pathway refers to the biological pathway that includes the activities of p73 and DCP l ⁇ and the associated proteins involved in p73 stability, p73 expression, p73 transcriptional regulation, DCP l ⁇ expression, DCP l ⁇ activity, mRNA decapping, and cytokine mRNA stability.
  • FIG. 1 Loss of TAp73 During the Innate Immune Response a, Analysis of TAp73 stability in wild type mice during challenge of airways with H. influenzae. Top panels, control and H. in/Zwenz ⁇ e-challenged nasal sinuses showing infiltration of neutrophils in the challenged mice (black arrow). Lower panels, TAp73 immunohistochemistry showing decrease in expression in the challenged mice. Green arrows, nuclei of ciliated epithelia. b, Western blot on airway epithelia of three wild type and four //.
  • FIG. 3 U251 CeUs Recapitulated Innate Immune Signaling Involving TAp73 a, Immunolocalization of NF- ⁇ B and TAp73 in the U251 glioblastoma cell line. Top, Control cells showing cytoplasmic NF- ⁇ B and nuclear TAp73. Middle and bottom panels show distribution of NF- ⁇ B and TAp73 8hr after exposure to H. influenzae or LPS, respectively, b, Western blot of U251 cells after exposure to H.
  • influenzae for the indicated times with or without the proteasome inhibitor MGl 32 using antibodies to TAp73 and nuclear lamin proteins
  • c Western blot of U251 cells treated with LPS for the indicated times using antibodies to TAp73 and nuclear lamin proteins
  • d RT-PCR analysis of inflammatory cytokine messages in U251 cells exposed to H. influenzae for the indicated times
  • e Quantitative PCR analysis of IL-8 mRNA in control and p73 shRNA expressing U251 cells 8hr after exposure to H. influenzae of the indicated titers.
  • FIG. 4 p73 Chromatin Immunoprecipitation Reveals Dcpl ⁇ as Direct Target a, Schematic diagram of p73 chromatin immunoprecipitation from U251 cells, b, Quantitative PCR analysis of p73 binding enrichment at p21 and Dcpl ⁇ genes, relative to a negative control (non-enriched) region c, Schematic of S. cerevisiae Dcpl protein and the two human orthologs, Dcpl ⁇ and Dq? 1 ⁇ . Shaded areas are regions of high homology.
  • Regions recognized by anti-Dcpl ⁇ monoclonal antibodies are designated by antibody icons, d, Anti-Dcpl ⁇ immunofluorescence on sections through nasal sinuses of wild type and p73-null P3 animals showing epithelial cell staining.
  • FIG. 5 Dcpl ⁇ Dynamics and Cytokine Expression a, Left, Quantitative PCR analysis of Dcpl ⁇ , TNF- ⁇ , and IL-8 mRNAs in U251 cells following exposure to H. influenzae bacteria. Right, Quantitative PCR analysis of Dcpl ⁇ , TNF- ⁇ , and IL-8 following treatment of U251 cells to LPS (500ng/ml). b, Left, Quantitative PCR analysis of IL-8 mRNA in U251 cell lines expressing control and Dcpl ⁇ -directed shRNAs following treatment with LPS (lOOng/ml).
  • TAp73 a nuclear transcription factor
  • Pathogens trigger the expression of inflammatory cytokine genes in ciliated cells as well as the degradation of TAp73.
  • TAp73 suppresses this inflammatory response by activating DCPl ⁇ , a gene encoding an mRNA decapping protein that is also highly expressed in ciliated airway epithelia.
  • Dcpl ⁇ may limit the expression of proinflammatory cytokine genes.
  • TAp73 and mRNA decapping mechanisms contribute to the dampening of such hyperinflammatory conditions, they are useful targets for the mitigation of acute and chronic airway disease. Methods for screening for agents that modulate the activity of these targets and therapeutic methods are described herein.
  • test agents can be used to evaluate test agents for their ability to modulate inflammation and other aspects of the innate immune response.
  • exemplary test agents include test compounds and gene therapy agents.
  • a test agent can be evaluated for its effect on the p73-DCPl ⁇ pathway, e.g., for its ability to increase p73-DCPl ⁇ pathway activity or for its ability to interact with or modulate a p73-DCPl ⁇ pathway.
  • Methods include in vitro and in vivo assays. Interactions include, for example, binding a target component, altering a covalent bond in a target component, or altering a biological or physiological function of a target compound (e.g., altering production, stability, or degradation of a target component).
  • a test compound or other agent that modulates the p73-DCPl ⁇ pathway can be prepared as a pharmaceutical composition and administered to a subject.
  • the test compounds can be obtained, for example, from a combinatorial library.
  • Some exemplary libraries include: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the one-bead/one-compound library method; and synthetic library methods using affinity chromatography selection.
  • These approaches can be used, for example, to produce peptide, non-peptide oligomer or small molecule libraries of compounds (see, e.g., Lam (1997) Anticancer Drug Des. 12:145).
  • a high throughput screening approach to a library of test compounds includes one or more assays, e.g., a combination of assays. Information from each assay can be stored in a database, e.g., to identify candidate compounds that can serve as leads for optimized or improved compounds, and to identify SARs.
  • a biological library includes polymers that can be encoded by nucleic acid.
  • Such encoded polymers include polypeptides and functional nucleic acids (such as nucleic acid aptamers (DNA, RNA), double stranded RNAs (e.g., RNAi), ribozymes, and so forth).
  • the biological libraries and non-biological libraries can be used to generate peptide libraries.
  • Another example of a biological library is a library of dsRNAs (e.g., siRNAs), or precursors thereof.
  • a library of nucleic acids that can be processed or transcribed to produce double-stranded RNAs (e.g., siRNAs)) is also featured.
  • Cell-based assays can be used to evaluate a test agent.
  • the cell for example, can be of mammalian origin, (e.g., from a human, a mouse, rat, primate, or other non-human).
  • the method can include contacting a test agent to the cell and evaluating p73 expression, or activity, DCP l ⁇ expression or activity, or cytokine expression or activity.
  • Evaluating expression can include one or more evaluating mRNA levels and evaluating protein levels.
  • the cell includes a reporter gene that has a DCP l ⁇ transcriptional regulatory sequence operably linked to a sequence encoding a detectable protein, e.g., a fluorescent protein (such as GFP) or a detectable enzyme (such as LacZ or CAT).
  • a detectable protein e.g., a fluorescent protein (such as GFP) or a detectable enzyme (such as LacZ or CAT).
  • Non-human animals can be used to evaluate a test compound.
  • the test agent is evaluated in a mouse lacking the p73 gene.
  • the test agent can be evaluated for its ability to ameliorate one or more phenotypes of the p73 deficient mouse, e.g., airway inflammation in the mouse.
  • Exemplary p73 proteins are described in Kaghad et al, Cell 90:809-819 (1997). p73 proteins include a DNA binding domain that is homologous to that of p53. The ability of a protein to have p73 DNA binding activity can be determined, e.g., by comparing DNA binding ability in vitro to p73. An exemplary assay is described in Lokshin et al. Nucleic Acids Res. 2007;35(l):340-52. p73 availability is regulated in part by the proteasome.
  • Proteasome activity can be reduced using inhibitors, such as lactacystin, MGl 32, CEP-8770(Cell Therapeutics, Inc.), CEP- 18770 (Cephalon), and NPI-0052 (Nereus).
  • inhibitors such as lactacystin, MGl 32, CEP-8770(Cell Therapeutics, Inc.), CEP- 18770 (Cephalon), and NPI-0052 (Nereus).
  • lactacystin analogs can be used, such as those described in US Pat. 6838477.
  • Other inhibitors can be identified, e.g., using the screening methods described herein.
  • Exemplary p73 proteins include those proteins having one or more biological functions of p73, e.g., the ability to increase DCPl ⁇ expression. These proteins are said to have a p73 activity. Such proteins include those proteins that are at least 85, 90, 95, 96, 97, 98, or 99% identical to human p73, e.g., in the DNA binding domain or throughout the amino acid sequence of TAp73. Biological function can be evaluated, for example, by expressing a particular protein in a cell lacking p73, e.g., a cell obtained from a mouse lacking p73.
  • Dcpl ⁇ Activation of Dcpl ⁇ by TAp73 may be sufficient to attenuate the overall inflammatory response.
  • An exemplary Dcpl ⁇ sequence is described at NCBI Accession No. NP_689853 (MoI. Cell. Biol. 22 (23), 81 14-8121 (2002)).
  • Dcpl ⁇ mediates decapping and hence inactivation of mRNAs. Decapping and overall message decay occur in common cytoplasmic organelles or "P bodies" 40> , which may contribute to the specificity and efficiency of the decapping process.
  • RNA endonuclease machinery that targets clusters of AU-rich (AUR) sequences found in the 3 '-untranslated regions of cytokine messages 34 ' 38 - 46"48 .
  • AUR AU-rich
  • the complexes that bind the AUR sites also interact with the decapping proteins, suggesting that the AUR sequences may dictate not only the de-polyadenylation activity but the specificity of Dcpl ⁇ -dependent decapping activity in the airways 21 ' 35 ' 42 .
  • Artificial transcription factors can also be used to regulate expression of TAp73 or DCP l ⁇ .
  • the artificial transcription factor can be designed or selected from a library, e.g., for ability to bind to a sequence in a regulatory region, e.g., the promoter, of the gene to be regulator.
  • the transcription factor may be selected for ability to bind the region of the DCP l ⁇ promoter that is bound by Tap73.
  • the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods En ⁇ ymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol 19:656; and Wu et al. (1995) Proc. Natl. Acad.
  • an artificial transcription factor can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription.
  • the artificial transcription factor can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., U.S. Pat. No. 6,534,261).
  • the heterologous nucleic acid that includes a sequence encoding the artificial transcription factor can be operably linked to an inducible promoter, e.g., to enable fine control of the level of the artificial transcription factor in the cell, e.g., an airway cell, e.g., a ciliated airway cell.
  • an inducible promoter e.g., to enable fine control of the level of the artificial transcription factor in the cell, e.g., an airway cell, e.g., a ciliated airway cell.
  • any suitable delivery vehicle can be used to deliver a nucleic acid into a cell, e.g., an airway cell.
  • exemplary delivery vehicles are viral packages.
  • nucleic acids that can be delivered include nucleic acid expression vectors that include sequences that encode proteins that modulate the p73-DCPl ⁇ pathway, e.g., proteins having p73 or DCP l ⁇ activity.
  • examples of such vectors include replication defective retroviral vectors, adenoviral vectors and adeno-associated viral vectors (AAVs).
  • Exemplary methods for the preparation and use of viral vectors are described in WO 96/13597, WO 96/33281, WO 97/15679, and Trapnell et al., Curr. Opin. Biotechnol. 5(6):617-625, 1994.
  • Adeno-associated virus is a nonpathogenic human parvovirus, capable of site- specific integration into chromosome 19. Exemplary methods for the preparation and use of AAVs are described in Fisher et al., Nature Medicine 3(3):306-312, 1997; Xiao et al., Jo. Virol. 70(11): 8098-8108, 1996; Kaplitt et al., Ann. Thorac. Surg. 62: 1669-1676, 1996.
  • coding sequences can be controlled by transcriptional regulatory sequences, e.g., promoters and enhancers, that are specific for a particular cell type, e.g., ciliated cells.
  • transcriptional regulatory sequences e.g., promoters and enhancers
  • exemplary promoters include: those of Foxo-Jl and dynein light chain.
  • disorders that can be treated by the agents described herein include chronic airway diseases, including asthma, rhinitis, bronchitis, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). They can be used to treat respiratory disorders caused by infectious agents, such as viruses (e.g., cold and flu viruses, respiratory syncytial virus, paramyxovirus, rhinovirus and influenza viruses).
  • infectious agents such as viruses (e.g., cold and flu viruses, respiratory syncytial virus, paramyxovirus, rhinovirus and influenza viruses).
  • the methods are useful for asthmas associated with or provoked by non- biological agents, e.g., smoke exposure (e.g., cigarette-induced and industrial smoke), as well as industrial and occupational exposures, such as smoke, ozone, noxious gases, sulfur dioxide, nitrous oxide, fumes, including isocyanates, from paint, plastics, polyurethanes, varnishes, etc., wood, plant or other organic dusts, etc.
  • smoke exposure e.g., cigarette-induced and industrial smoke
  • industrial and occupational exposures such as smoke, ozone, noxious gases, sulfur dioxide, nitrous oxide, fumes, including isocyanates, from paint, plastics, polyurethanes, varnishes, etc., wood, plant or other organic dusts, etc.
  • the methods are also useful for asthmatic incidents associated with food additives, preservatives or pharmacological agents.
  • methods for treating, inhibiting or alleviating the types of asthma referred to as silent asthma or cough variant asthma.
  • the agents described herein can be used in vitro, ex vivo, or in vivo. They can be incorporated into a pharmaceutical composition, e.g., by combining the agent with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition may contain, in addition to the agent and carrier, one or more of: various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials.
  • Pharmaceutically acceptable materials is generally a nontoxic material that does not interfere with the effectiveness of the biological activity of the agent.
  • the characteristics of the carrier can depend on the route of administration.
  • the pharmaceutical composition described herein may be in the form of a liposome that includes the agent, e.g., in addition to pharmaceutically acceptable carriers, with amphipathic agents such as lipids that exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers while in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
  • Exemplary methods for preparing such liposomal formulations include methods described in U.S. Pat. Nos. 4,235,871 ; 4,501,728; 4,837,028; and 4,737,323.
  • a pharmaceutical composition can be administered in a therapeutically effective amount, e.g., an amount sufficient to cause a meaningful patient benefit, e.g., amelioration of symptoms of, healing of, or increase in rate of healing of such conditions, in a typical patient.
  • the amount can be determined by a physician, e.g., based on evaluating a patient and patient parameters, e.g., weight, gender, age, and so forth.
  • the amount of the pharmaceutical composition can depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone.
  • the pharmaceutical composition can be administered to normal patients or patients who do not show symptoms, e.g., in a prophylactic mode.
  • An attending physician may decide the amount with which to treat each individual patient. For example, an attending physician can administer low doses of the agent and observe the patient's response. Larger doses of the agent may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further.
  • a composition may contain between about 0.001 mg to 5 mg of a small molecule agent.
  • the composition can be administered in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection.
  • the composition of a composition for parenteral administration can be adapted in view factors such as pH, isotonic! ty, stability, and the like, e.g., to optimize the composition for physiological conditions, binding agent stability, and so forth.
  • a pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection can contain, e.g., an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants, or other additive.
  • a composition that includes an agent described herein can be formulated for inhalation or other mode of pulmonary delivery. Accordingly, the agent can be administered by inhalation to airway tissue.
  • the agent is formulated for a nebulizer.
  • the agent can be stored in a lyophilized form (e.g., at room temperature) and reconstituted in solution prior to inhalation. It is also possible to formulate the agent for inhalation using a medical device, e.g., an inhaler. See, e.g., U.S. Pat. No. 6,102,035 (a powder inhaler) and U.S. Pat. No. 6,012,454 (a dry powder inhaler).
  • the inhaler can include separate compartments for the agent at a pH suitable for storage and another compartment for a neutralizing buffer and a mechanism for combining the agent with a neutralizing buffer immediately prior to atomization.
  • the inhaler is a metered dose inhaler.
  • the three common systems can be used to deliver drugs locally to the pulmonary air passages include dry powder inhalers (DPIs), metered dose inhalers (MDIs) and nebulizers.
  • MDIs the most popular method of inhalation administration, may be used to deliver medicaments in a solubilized form or as a dispersion.
  • MDIs comprise a Freon or other relatively high vapor pressure propellant that forces aerosolized medication into the respiratory tract upon activation of the device.
  • DPIs generally rely entirely on the inspiratory efforts of the patient to introduce a medicament in a dry powder form to the lungs.
  • Nebulizers form a medicament aerosol to be inhaled by imparting energy to a liquid solution.
  • the agent is delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant or a nebulizer.
  • the agent may be in the form of a dry particle or as a liquid.
  • Particles that include the agent can be prepared, e.g., by spray drying, by drying an aqueous solution of the agent with a charge neutralizing agent and then creating particles from the dried powder or by drying an aqueous solution in an organic modifier and then creating particles from the dried powder.
  • the agent may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the agent ⁇ and a suitable powder base such as lactose or starch, if the particle is a formulated particle.
  • formulated or unformulated compound in addition to the formulated or unformulated compound, other materials such as 100% DPPC or other surfactants can be mixed with the agent to promote the delivery and dispersion of formulated or unformulated compound. Methods of preparing dry particles are described, for example, in WO 02/32406. Delivery enhancers such as surfactants can be used to further enhance pulmonary delivery.
  • Surfactants include but are not limited to phosphoglycerides, e.g., phosphatidylcholines, L-alpha-phosphatidylcholine dipalmitoyl (DPPC) and diphosphatidyl glycerol (DPPG); hexadecanol; fatty acids; polyethylene glycol (PEG); polyoxyethylene-9--; auryl ether; palmitic acid; oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; poloxomer; sorbitan fatty acid ester; sorbitan trioleate; tyloxapol; and phospholipids.
  • phosphoglycerides e.g., phosphatidylcholines, L-alpha-phosphatidylcholine dipalmitoyl (DPPC) and diphosphatidyl glycerol (DPPG); hexadecanol; fatty acids; polyethylene glycol (PEG); polyoxyethylene-9
  • the methods described herein can employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook. Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B.
  • the innate immune system employs sets of highly conserved receptors to detect and signal the presence of pathologic microorganisms 4"8 .
  • These receptors including Toll-like receptors (TLR) for lipopolycaccharide (LPS) of Gram-negative bacteria and peptidoglycans of Gram-positive bacteria, and NOD1/CARD4 for intracellular pathogens, are found in a wide variety of cells. Activation of these receptors drives the maturation of professional immune cells such as macrophages and dendritic cells, which in turn act in the MHC-dependent activation of T cells to promote the evolution of asthma 9 ' 10 .
  • TLR Toll-like receptors
  • LPS lipopolycaccharide
  • NOD1/CARD4 for intracellular pathogens
  • Ciliated epithelial cells the predominant cell type of upper airway surfaces- act as mechanical drivers for extracellular mucus flow 12 ' 13 .
  • These airway epithelia cells express high levels of certain 7b//-like receptors, are capable of inflammatory cytokine production, and potentially offer the largest surface area for pathogen detection in the upper airways and nasal passages 7> 14 ⁇ 18 .
  • the data disclosed herein indicate that p73 can mediated response through the mammalian ortholog of the yeast mRNA decapping protein Dcpl ⁇ , to regulate the inflammatory response via the control of cytokine message stability.
  • the p73-null mouse develops profound neonatal rhinitis and otitis media marked by dense infiltration of neutrophils .
  • An examination of these mice over time revealed the chronic nature of the rhinitis from neonates through adulthood (Fig. Ia).
  • Inspection of the entire airway revealed a generalized inflammation marked by neutrophil infiltration of the nasal passages, trachea, and pulmonary bronchi (Fig. Ia; Supplementary Fig. Ia).
  • This inflammation was not mitigated by pre- and post-natal treatment with broad-spectrum antibiotics (Supplementary Fig. Ib).
  • these findings suggest a hyperinfiammatory condition rather than a normal response to pathogen challenge.
  • cytokines such as KC, MIP-2, and TNF- ⁇
  • KC KC
  • MIP-2 inflammatory cytokines
  • TNF- ⁇ inflammatory cytokines
  • LIX expression was obvious in p73-null tissues or those exposed to LPS or pathogenic bacteria (Fig. 2c), consistent with the notion that LIX is produced locally by epithelial cells.
  • Fig. 2d To further probe the apparent loss of p73 following exposure to pathogenic bacteria, we employed an in vitro preparation of the trachea 27 , whose luminal surface is densely lined with ciliated epithelial cells (Fig. 2d). After 12 hours of challenge by H. influenzae in vitro, TAp73 expression was also muted in the trachea (Fig. 2e,f). This loss of TAp73 is blocked by MGl 32, an inhibitor of the proteasome 28 . Together, these in vivo models indicate that p73 acts to suppress the inflammatory response and is targeted for destruction during the normal course of innate immune response activation.
  • U251 Glioblastoma Cells as Model of p73-Regulated Innate Immune Response p73 expression is apparent in a number of relatively common, transformed lines 19 .
  • U251 cells derived from a human glioblastoma, expresses both TAp73 and the NF- ⁇ B transcription factor, a key transducer of innate immune signaling from Toll-like receptors 29"30 .
  • TAp73 Controls a Gene Involved in Messenger RNA Stability
  • a potential mechanism for TAp73's action in the innate immune response is the direct repression of inflammatory cytokine genes.
  • We tested this possibility by examining overlapping regions of the IL-8 gene for binding of TAp73 in chromatin immunoprecipitations of U251 cells using anti-TAp73 antibodies. None of the many DNA elements implicated in the control of IL-8 34 expression showed enhanced representation in the TAp73 chromatin immunoprecipitations.
  • To obtain a more global view of direct transcriptional targets of TAp73 in U251 cells we hybridized DNA associated with TAp73 chromatin immunoprecipitations to oligonucleotide microarrays representing promoter regions of all known genes 35 .
  • Dcpl ⁇ was amongst the strongest positives we have recorded in the analysis of p53, p63, or p73 binding sites, with an average occupancy scores of 330-fold over background 35 ' 39 .
  • tissue distribution of Dcpl ⁇ we generated monoclonal antibodies to the N-terminal domains of human Dcpl ⁇ that are the most highly conserved with yeast Dcpl (Fig. 4c).
  • Dcpl ⁇ is under the control of TAp73, this might be reflected in the relative responses to H. influenzae and LPS, which have differential effects on TAp73 stability.
  • U251 cells treated with LPS showed only a modest decline in Dcpl ⁇ transcript levels and IL-8 and TNF- ⁇ transcript levels peaked at 7 and 4 hours, respectively, and declined thereafter.
  • Ciliated epithelial cells are key components of the innate immune response in the airways and p73 is a prominent regulator of this response. These cells are able to detect and respond to pathogens. Accordingly, controlling these cells can be used to both address infections and to modulate acute and chronic airway inflammation.
  • TAp73 appears to establish a buffer against hyperinflammatory responses by establishing a threshold for cytokine activation and limiting the overall lifespan of cytokine messenger RNAs.
  • the need for this TAp73 -dependent buffer to the innate immune response by ciliated airway cells might be dictated by the abundance of pathogenic and innocuous microorganisms in the respiratory passages. Thus cells in the epithelial lining might have to temper an inflammatory response until they can distinguish between hapless pathogens encountered in the mucociliary flow and those attempting to mount colonization and infection.
  • the p73-null mouse BALB/c strain was generated as previously described 20 .
  • Sulfatrim a broad-spectrum antibiotic, was added to the drinking water (5 ml per 200 ml) of pregnant mice at 18.5 days p.c. in experiments involving antibiotics.
  • Monoclonal antibodies to p73 were generated by immunizing six-week old mice with the C-terminal portion (amino acids 401-636) or the transactivation domain (TA; amino acids 1-95) of human TAp73.
  • mice were immunized with the N-terminal 200 amino acids of human Dcpl ⁇ .
  • Spleen cells were fused with the myeloma line NSO and hybridoma supernatants screened by immunofluorescence of BHK cells transfected with either p73 or Dcpl ⁇ . Positive clones were subsequently subcloned and re-screened for stable antibody production.
  • Haeomophilus influenzae type b, was purchased from the ATCC (49247) and cultured according to ATCC protocols. Newborn mice used in experiments were from pregnant CFW outbred mice purchased from Charles River Laboratories (Wilmington, MA). Mice received intranasally either 3 ⁇ l saline or 3 ⁇ l saline containing 1 x 10 5 colony forming units of H. influenzae every four hours. The mice were sacrificed after 24 hours for either tissue harvesting or for fixation for histology examination.
  • Dissected mouse tissues were homogenized on ice in 50 mM TRIS, pH 8.0, 150 mM NaCl, 2 mM MgCl 2 , 0.5% Triton X-100, 0.5 mM DTT, and Ix protease inhibitor cocktail (Roche).
  • Supernatants were recovered after spinning at 15,000 x g at 4 0 C for 5 min.
  • the supernatants were mixed with an equal volume of 2 x Laemmli sample buffer and 10OmM ⁇ -mercaptoethanol and boiled at 95 0 C for 5 min. All samples were spun for 5 min at 15,000 x g before loading onto SDS-PAGE gels.
  • RNA samples were derived from p73-null mice and their wild type littermates that were sacrificed at El 8.5 and P3.5.
  • Total RNA was extracted using TRIzol reagent (Invitrogen).
  • First strand cDNA was synthesized using the Superscript first-strand synthesis system for RT-PCR kit (Invitrogen).
  • a standard RT-PCR was run to examine changes in RNA expression for the proinflammatory cytokines TNF- ⁇ , KC, MIP -2 and LIX.
  • PCR amplifications were performed using 29 cycles in a total volume of 25 ⁇ l containing 1 ⁇ l of the cDNA sample (derived from 20 ng of total RNA). For examining ⁇ -tubulin or ⁇ -actin expression, similar reaction conditions but 26-27 cycles were used.
  • Quantitative PCR was performed with Applied BioSystems methods using the Universal PCR Master Mix (no AmpErase UNG (4323018) and TaqMan Gene Expression primers GAPDH (Hs99999905), IL-8 (Hs00174103), TNF- ⁇ (Hs00174128), and Dcpl ⁇ (Hs00398931). Standard qPCR reactions were performed according to Applied BioSystem protocols in a total volume of 20 ⁇ l containing 20-50 ng cDNA.
  • Chromatin immunoprecipitation (ChIP)
  • U251 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum. Trypsinized cells were placed into fresh media and cross-linked by the addition of formaldehyde to a final concentration of 1% for 8 minutes. Formaldehyde was quenched by adding glycine to a final concentration of 0.2M. Cells were then spun down and washed 2 times in cold IX phosphate buffered saline (PBS), followed by 3 washes with Cell Lysis Buffer (10 mM Tris-Cl pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.5% NP-40, protease inhibitors).
  • PBS cold IX phosphate buffered saline
  • the cell pellet was then resuspended in Sonication buffer (10 mM Tris-Cl pH 7.5, 200 mM NaCl, 3 mM MgCl 2 , 4% NP-40, 1% SDS, protease inhibitors) and sonicated with a Branson Sonifier for 5 X 30 sec minute pulses at setting #5, 60% output.
  • Sonication buffer (10 mM Tris-Cl pH 7.5, 200 mM NaCl, 3 mM MgCl 2 , 4% NP-40, 1% SDS, protease inhibitors
  • the chromatin was then spun down at 14 x g for 30 minutes and the supernatant was diluted 5-fold with IP-dilution buffer (20 mM Tris-Cl pH 8, 2 mM EDTA, 150 mM NaCl, *% TritonX-100, protease inhibitors).
  • Diluted chromatin was precleared with protein A/G Sepharose beads, and a portion reserved for control, or 'input', DNA (i.e. omitting immunoprecipitation). The remaining chromatin was incubated with a cocktail of three different monoclonal antibodies to p73 (1 ICl 2, 3A6, and 10H7) coupled to protein A/G sepharose beads overnight at 4 0 C.
  • Beads were then washed in the following: IX with IP dilution buffer; 2X Wash Buffer #1 (20 mM Tris-Cl pH 8, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS); 2X Wash Buffer #2 (20 mM Tris-Cl pH 8, 2 mM EDTA, 500 mM NaCl, 1% TritonX-100); 2 X Wash Buffer #3 ((10 mM Tris-Cl pH 8, 1 mM EDTA, 0.25 M LiCl; 1% NP-40, 1 % deoxycholate); 2 X 10 mM Tris-Cl pH 8, 1 mM EDTA.
  • 2X Wash Buffer #1 (20 mM Tris-Cl pH 8, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS
  • 2X Wash Buffer #2 (20 mM Tris-Cl pH 8, 2 mM
  • qPCR was performed essentially as described 35 , using an Applied Biosystems 7300 sequence detector for SYBR green fluorescence.
  • the PCR program was: 95 0 C 10 min, followed by 40 cycles of 95 0 C, 30 sec, 60 0 C, 45 sec; 72 0 C, 1 min.
  • Fold enrichment for a genomic region was determined relative to a non-enriched region (exon 3 of the histone H3 gene).
  • For each site we calculated the occupancy units defined as the fold enrichment value minus background (H3 reference value set to 1).
  • mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404, 99-103 (2000).
  • TLR4 Toll-like receptor 4

Abstract

L'augmentation de l'activité de la voie p73/DCP1ß dans les cellules des voies respiratoires du sujet peut servir dans un procédé d'inhibition d'une inflammation des voies respiratoires chez un sujet. L'activité de la voie p73/DCP1ß peut être augmentée de toute une diversité de manières, telles que la réduction de la dégradation de p73, l'augmentation des taux ou de l'activité de p73 et l'augmentation de l'activité de DCP1ß.
PCT/US2008/065951 2007-06-05 2008-06-05 Modulation d'une inflammation des voies respiratoires WO2008151289A1 (fr)

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US20050196780A1 (en) * 2000-09-19 2005-09-08 University Of Medicine And Dentistry Of New Jersey Compositions and methods for reproducing and modulating mammalian messenger RNA decapping
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