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WO2014082993A2 - Cibles moléculaires et composés, et procédés d'identification de ceux-ci, utiles dans la régulation à la baisse de la réponse th2 - Google Patents

Cibles moléculaires et composés, et procédés d'identification de ceux-ci, utiles dans la régulation à la baisse de la réponse th2 Download PDF

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Publication number
WO2014082993A2
WO2014082993A2 PCT/EP2013/074723 EP2013074723W WO2014082993A2 WO 2014082993 A2 WO2014082993 A2 WO 2014082993A2 EP 2013074723 W EP2013074723 W EP 2013074723W WO 2014082993 A2 WO2014082993 A2 WO 2014082993A2
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Prior art keywords
compound
polypeptide
response
cell
target
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PCT/EP2013/074723
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English (en)
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WO2014082993A3 (fr
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Richard JANSSEN
Jamil AARBIOU
Annemarie LEKKERKERKER
Nick Vandeghinste
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Galapagos Nv
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Publication of WO2014082993A2 publication Critical patent/WO2014082993A2/fr
Publication of WO2014082993A3 publication Critical patent/WO2014082993A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • the present invention is in the field of molecular biology and biochemistry.
  • the present invention relates to methods for identifying agents capable of inhibiting the T h 2 response, in particular, agents that down-regulate T h 2 response in an individual. Inhibition of these processes is useful in the prevention and / or treatment of inflammatory disorders and other disorders involving an imbalance in T h l/T h 2 inflammatory responses.
  • the present invention provides methods for identifying agents for use in the prevention and / or treatment of asthma, allergic rhinitis, atopic dermatitis, fibrosis, atherosclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, glomerulonephritis, cryoglobulinemia, immune disorders or deficiency, rhinoconjunctivitis, anaphylaxis and/or cancers.
  • Cytokines are the messengers responsible for many signaling processes of the immune system, such as cell mediated immunity and allergic type responses. Although they are numerous, cytokines can be functionally divided into two groups: those that are pro-inflammatory and those that are essentially antiinflammatory but that promote allergic responses.
  • T cells are one of the major sources of cytokines. These cells possess specific receptors on their cell surface to allow recognition of foreign pathogens (e.g. bacterial or viral pathogens). They can also contribute to pathological conditions by recognizing normal tissue during episodes of autoimmune diseases.
  • pathogens e.g. bacterial or viral pathogens.
  • CD4 and CD8 cell surface molecules
  • T lymphocytes expressing CD4 are also known as helper T cells, and these are regarded as being the most prolific cytokine producers.
  • T h l Type 1 T helper
  • T h 2 Type 2 T helper
  • T h l-type cytokines tend to produce the pro-inflammatory responses. They produce Interferon gamma (IFNy), interleukin (IL)-2, and tumor necrosis factor (TNF ), which activate macrophages and are responsible for cell-mediated immunity and phagocyte-dependent protective responses. Excessive proinflammatory responses can lead to uncontrolled tissue damage, so there needs to be a mechanism to counteract this.
  • the T h 2-type cytokines include interleukins 4 (IL4), 5(IL5), and 13 (IL13), which are associated with the promotion of IgE and eosinophilic responses in atopy, and also interleukin- 10 (IL10), which has more of an anti-inflammatory response.
  • T h 2 responses will counteract T h l mediated microbicidal action.
  • Polarized T h l and T h 2 cells not only exhibit different functional properties, but also show the preferential expression of some activation markers and distinct transcription factors (Rengarajan 2000). The ideal situation would therefore seem to be that humans should produce a well balanced T h l and T h 2 response, suited to the immune challenge.
  • the imbalance in those responses may lead to some severe conditions like allergic reactions and asthma. Many researchers regard allergy as a T h 2 weighted imbalance, and recently there have been attempts to identify ways to redirect allergic T h 2 responses in favor of T h l responses.
  • Epithelial cells such as those lining the intestine (IECs), release a number of mediators in response to triggers of allergic reaction and they contribute to the activation of dendritic cells (DCs) and subsequent T cell polarization into T h 2 cytokine-producing phenotype.
  • DCs dendritic cells
  • Those factors include thymic stromal lymphopoietin TSLP, granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostaglandin E2 (PGE2).
  • TSLP an interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses, which ultimately trigger T cells to adopt the T h 2 phenotype.
  • TSLP is a critical and essential factor for allergic inflammation
  • Wang 2006 Ziegler 2006
  • TSLP treatment of DCs leads to their functional maturation, and na ' ive CD4+ T cells that are targeted by these DCs obtain T h 2 phenotype, producing the T h 2 cytokines (Soumelis 2002).
  • T h 2 cells in turn recruit eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa.
  • These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma.
  • TSLP expression levels are increased in the lesional skin of atopic dermatitis patients (Soumelis 2002) and in the lungs of asthmatics (Ying 2005).
  • TSLP intestinal epithelial cells
  • Conditions like ulcerative colitis also appear to involve inflammatory T h 2 responses.
  • an inflammatory T h 2 condition in the mucosal lesions of UC patients may trigger increased TSLP expression by IECs, resulting in exacerbation of UC (Tanaka 2010).
  • TSLP was found to promote fibrosis as described in the US patent application US 2006- 0171943 (Wynn 2004).
  • general anti-inflammatory treatments are primarily used to treat fibrotic disorders. Such treatments are not always effective in reducing and preventing fibrosis (Wynn 2004). Therefore, there is a need remaining for developing treatments for reduction and prevention of fibrosis.
  • TSLP -neutralizing antibodies blocked the up-regulation of OX40L by DCs induced by breast cancer cells, thereby blocking their capacity to induce T h 2 cells in vitro.
  • Antibodies neutralizing TSLP, OX40L, or TSLPR inhibited tumor development and IL-13 secretion in the xenograft model.
  • protease activated receptor 2 (PAR2)-signaling is involved in TSLP regulation in cancer cells.
  • TSLP-driven inflammation might contribute to breast cancer pathogenesis and development via T h 2 polarization; and TSLP production by cancer cells is regulated via PAR2-signaling.
  • a similar role of TSLP was also demonstrated in pancreatic cancer (De Monte 2011). Therefore TSLP appears to play an important role in cancer development and blocking TSLP production may improve prognosis and treatment of different cancers.
  • the present invention provides genes and proteins (TARGETS) involved the signal transduction underlying the Th2 response, particularly in TSLP expression and function, which may be of benefit for the identification of new therapeutic interventions in these diseases.
  • TARGETS genes and proteins involved the signal transduction underlying the Th2 response, particularly in TSLP expression and function, which may be of benefit for the identification of new therapeutic interventions in these diseases.
  • the present invention is based on the discovery that agents that inhibit the expression and / or activity of the TARGETS disclosed herein are capable of down-regulating the type 2 T helper (T h 2) response, as indicated by a suppression of the release of cytokines from epithelial cells capable of activating T h 2 response, in particular a suppression of the release or expression of thymic stromal lymphopoietin (TSLP) and granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • T helper T helper
  • TSLP thymic stromal lymphopoietin
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the present invention therefore provides TARGETS which down-regulate T h 2 response, methods for screening for agents capable of down- regulating the expression and / or activity of TARGETS and the use of these agents in the prevention and / or treatment of diseases associated with an elevated T h 2 response.
  • the present invention provides TARGETS which are involved in or otherwise associated with T h 2-related disorders, in particular with T h 2 related inflammatory disorders and T h 2 related fibrotic disorders.
  • the present invention provides TARGETS which are involved in or otherwise associated with allergic inflammation, in particular, with asthma.
  • the present invention relates to a method for identifying compounds that are able to inhibit the T h 2 response, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36-70 (hereinafter "TARGETS”) and fragments thereof, under conditions that allow said polypeptide to bind to said compound, and measuring a binding affinity of the compound to said polypeptide.
  • a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36-70 (hereinafter "TARGETS”) and fragments thereof, under conditions that allow said polypeptide to bind to said compound, and measuring a binding affinity of the compound to said polypeptide.
  • the invention relates to a method for identifying compounds that are able to inhibit the T h 2 response, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36-70 (hereinafter "TARGETS") and fragments thereof, measuring the expression or activity of said polypeptide in the presence of the compound, and determining whether the expression or activity of said polypeptide is blocked, reduced or inhibited.
  • a compound that blocks, reduces or inhibits the expression or activity of a TARGET is a candidate activity-inhibitory agent.
  • it is further determined whether a biological property related to the down-regulation of T h 2 phenotype or of T h 2 response is altered, e.g.
  • the method relates to identifying compounds which modulate T h l/T h 2 balance.
  • aspects of the present method include the in vitro assay of compounds using a TARGET polypeptide, or fragments thereof, and cellular assays wherein TARGET polypeptide inhibition is followed by observing indicators of efficacy including, for example, TARGET expression levels and/or TARGET enzymatic activity, expression or release of cytokines capable of inducing T h 2 response or other biological properties related to the suppression of T h 2 activation.
  • the present invention further provides a method for identifying a compound that down-regulates T h 2 response, said method comprising contacting a compound with a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-35, determining the binding of the compound to the polynucleotide; and identifying the compound that down-regulates the T h 2 response.
  • a method for identifying a compound that down-regulates T h 2 response comprising contacting a compound with a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-35, determining the binding of the compound to the polynucleotide; and identifying the compound that down-regulates the T h 2 response.
  • Such method might be further performed in a cellular system or in a form of in vitro assay. Aspects of the method include a screening method when the target polynucleotide is an RNA
  • the present invention also relates to
  • expression inhibitory agents comprising a polynucleotide selected from the group of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said polynucleotide comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a TARGET polypeptide said polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO 1-35: and
  • compositions comprising said agent(s), useful in the treatment, or prevention, of conditions and disease related to elevated T h 2 response.
  • Another aspect of the invention is a method of treatment or prevention of elevated T h 2 response, in a subject suffering from, or susceptible to, a condition triggered by an elevated T h 2 response, particular by a T h l/T h 2 response imbalance, by administering a pharmaceutical composition comprising an effective TARGET-expression inhibiting amount of an expression-inhibitory agent or an effective TARGET activity inhibiting amount of an activity-inhibitory agent.
  • Another aspect of this invention relates to the use of agents which inhibit a TARGET as disclosed herein in a therapeutic method, a pharmaceutical composition, and the manufacture of such composition, useful for the treatment of diseases involving elevated T h 2 response, in particular, allergic inflammation.
  • Figure 1 shows a schematic overview of the assay set up used for primary screen
  • Figure 2 shows a typical plate layout used for primary screening.
  • Column 7 contains putative negative (A-C) and positive (D-H) controls.
  • Figure 3 presents Inter-quartile range (IQR) scores of sample and control duplicates of the primary screen
  • Figure 4 demonstrates a typical experimental layout of the rescreen
  • Figure 5 shows a typical experimental layout of the on-target screen
  • Figure 6 provides a general plate layout used in the on-target siRNA screening. Each plate contained 30% negative controls and 56 'test' siRNAs targeting 56 different genes (the 3 different siRNA versions for each gene were put on different plates).
  • Figure 7 shows data on TSLP secretion in normal human bronchial epithelial (NHBE) cells upon trigger with rhinovirus serotype 2 (RV2) and rhinovirus serotype 59 (RV59) with (+) or without (-) IL-4 at four dilutions (decreasing RV levels with decreasing color intensity).
  • RV2 rhinovirus serotype 2
  • RV59 rhinovirus serotype 59
  • Figure 8 demonstrates the effect of positive and negative control siRNAs on GM-CSF and TSLP secretion induced by RV59.
  • Figure 9 illustrates Z-scores of biological duplicate targets in the secondary assay
  • agent means any molecule, including polypeptides, polynucleotides, natural products and small molecules.
  • agent includes compounds such as test compounds or drug candidate compounds.
  • the term 'activity inhibitory agent' or 'activity inhibiting agent' means an agent, e.g. a polypeptide, small molecule, compound designed to interfere or capable of interfering selectively with the activity or expression of a specific polypeptide or protein normally expressed within a cell.
  • the term 'agonist' refers to an agent that stimulates the receptor the agent binds to in the broadest sense.
  • allergic inflammation refers to a family of conditions associated with T h 2 over-response and T h l/T h 2 imbalance leading to allergic symptoms. Specific examples of such conditions include allergic asthma and atopic dermatitis.
  • the term 'antagonist' is used to describe an agent that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses, or prevents or reduces agonist binding and, thereby, agonist-mediated responses.
  • the term 'assay' means any process used to measure a specific property of an agent, including a compound.
  • a 'screening assay' means a process used to characterize or select compounds based upon their activity from a collection of compounds.
  • binding affinity' is a property that describes how strongly two or more compounds associate with each other in a non-covalent relationship. Binding affinities can be characterized qualitatively, (such as 'strong', 'weak', 'high', or 'low') or quantitatively (such as measuring the KD).
  • the term 'carrier' means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition.
  • a carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution.
  • physiologically acceptable carriers include aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide
  • the term 'complex' means the entity created when two or more compounds bind to, contact, or associate with each other.
  • the term 'compound' is used herein in the context of a 'test compound' or a 'drug candidate compound' described in connection with the assays of the present invention.
  • these compounds comprise organic or inorganic compounds, derived synthetically or from natural sources.
  • the compounds include inorganic or organic compounds such as polynucleotides (e.g. siRNA or cDNA), lipids or hormone analogs.
  • Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, including polypeptide ligands, enzymes, receptors, channels, antibodies or antibody conjugates.
  • condition' or 'disease' means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (for example, biochemical indicators).
  • abnormal clinical indicators for example, biochemical indicators.
  • 'disease' refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators.
  • condition associated with a T h l/T h 2 imbalance means a set of conditions for which the T h 2 response or T h l response has become more dominant. Many conditions are associated with such imbalance, in particular, inflammatory disorders and diseases.
  • 'contact' or 'contacting' means bringing at least two moieties together, whether in an in vitro system or an in vivo system.
  • polypeptides relate to those peptides, oligopeptides, polypeptides, proteins and enzymes that comprise a stretch of contiguous amino acid residues of the polypeptide and that retain a biological activity of the protein, for example, polypeptides that have amino acid mutations compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • a derivative may further comprise additional naturally occurring, altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally occurring form of the polypeptide.
  • polynucleotide may also contain one or more non-amino acid substituents, or heterologous amino acid substituents, compared to the amino acid sequence of a naturally occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence.
  • the term 'derivatives of a polynucleotide' relates to DNA-molecules, RNA-molecules, and oligonucleotides that comprise a stretch of nucleic acid residues of the polynucleotide, for example, polynucleotides that may have nucleic acid mutations as compared to the nucleic acid sequence of a naturally occurring form of the polynucleotide.
  • a derivative may further comprise nucleic acids with modified backbones such as PNA, polysiloxane, and 2'-0-(2-methoxy) ethyl-phosphorothioate, non- naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.
  • nucleic acids with modified backbones such as PNA, polysiloxane, and 2'-0-(2-methoxy) ethyl-phosphorothioate, non- naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanu
  • the term 'endogenous' shall mean a material that a mammal naturally produces. Endogenous in reference to the term 'enzyme', 'protease', 'kinase', or G-Protein Coupled Receptor ('GPCR') shall mean that which is naturally produced by a mammal (for example, and not by limitation, a human). In contrast, the term non- endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not by limitation, a human). Both terms can be utilized to describe both in vivo and in vitro systems.
  • the endogenous or non-endogenous TARGET may be in reference to an in vitro screening system.
  • screening of a candidate compound by means of an in vivo system is feasible.
  • the term 'expressible nucleic acid means a nucleic acid coding for or capable of encoding a proteinaceous molecule, peptide or polypeptide, and may include an RNA molecule, or a DNA molecule.
  • the term 'expression' comprises both endogenous expression and non-endogenous expression, including overexpression by transduction.
  • 'expression inhibitory agent' or 'expression inhibiting agent' means an agent, e.g. a polynucleotide designed to interfere or capable of interfering selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. More particularly, 'expression inhibitory agent' comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 15-30, particularly at least 17, sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein. Exemplary expression inhibitory molecules include ribozymes, double stranded siRNA molecules, self-complementary single- stranded siRNA molecules, genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones.
  • fragment of a polynucleotide' relates to oligonucleotides that comprise a stretch of contiguous nucleic acid residues that exhibit substantially a similar, but not necessarily identical, activity as the complete sequence.
  • 'fragment' may refer to a oligonucleotide comprising a nucleic acid sequence of at least 5 nucleic acid residues (preferably, at least 10 nucleic acid residues, at least 15 nucleic acid residues, at least 20 nucleic acid residues, at least 25 nucleic acid residues, at least 40 nucleic acid residues, at least 50 nucleic acid residues, at least 60 nucleic residues, at least 70 nucleic acid residues, at least 80 nucleic acid residues, at least 90 nucleic acid residues, at least 100 nucleic acid residues, at least 125 nucleic acid residues, at least 150 nucleic acid residues, at least 175 nucleic acid residues, at least 200 nucleic acid residues, or at least 250 nucleic acid residues) of the nucleic acid sequence of said complete sequence.
  • nucleic acid residues preferably, at least 10 nucleic acid residues, at least 15 nucleic acid residues, at least 20 nucleic acid residues
  • the term 'fragment of a polypeptide' relates to peptides, oligopeptides, polypeptides, proteins, monomers, subunits and enzymes that comprise a stretch of contiguous amino acid residues, and exhibit substantially a similar, but not necessarily identical, functional or expression activity as the complete sequence.
  • 'fragment' may refer to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of said complete sequence.
  • amino acid residues preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues,
  • the term 'hybridization' means any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • the term 'hybridization complex' refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (for example, COt or ROt analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (for example, paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • stringent conditions refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent, for example, formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency.
  • standard hybridization conditions' refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash. However, one skilled in the art will appreciate that such 'standard hybridization conditions' are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like.
  • Standard hybridization conditions are whether the two sequences hybridizing are RNA-RNA, DNA- DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20NC below the predicted or determined Tm with washes of higher stringency, if desired.
  • the term 'inflammatory disorder' refers to any disease or disorder characterized by local inflammation at a site of injury or infection and includes, without limitation, allergic inflammation, autoimmune diseases, and disorders characterized by undesired immune cell accumulation at a local tissue site.
  • the term 'inhibit' or 'inhibiting', in relationship to the term 'response' means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
  • the term 'inhibition' refers to the reduction, down regulation of a process or the elimination of a stimulus for a process, which results in the absence or minimization of the expression or activity of a protein or polypeptide.
  • immunostomulant refers to the substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components.
  • immunostomulant might be a single molecule, combination of different molecules, or parts of pathogenic organisms like bacteria and viruses.
  • immunostimulant is polyinosinic:polycytidylic acid (Poly I:C).
  • Poly I:C can be used in combination with IL4 or without it.
  • the term 'induction' refers to the inducing, up-regulation, or stimulation of a process, which results in the expression, enhanced expression, activity, or increased activity of a protein or polypeptide.
  • 'ligand' means an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
  • downstream-regulator of T h 2 response refers to any agent capable of down-regulating or otherwise inhibiting the processes and pathways involved in conversion of naive T cells into T h 2 phenotype.
  • such molecules might be cytokines or other signalling molecules released by cells, which in turn affect the pathways involved in differentiation of naive T cells into T h 2 phenotype.
  • such regulators might be small molecule compounds that down-regulate the activity or expression of gene/proteins that stimulate the differentiation of naive T cells into the T h 2 phenotype.
  • the term "down-regulation of T h 2 response" is used in the context of the down- regulation of processes responsible for the differentiation of naive T cells into the T h 2 phenotype.
  • the required extent of the effect of the down-regulator of T h 2 response may vary. In particular cases a small decrease in expression of a particular gene might lead to a significant down-regulation of T h 2 response.
  • salts refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds which inhibit the expression or activity of TARGETS as disclosed herein. These salts can be prepared in situ during the final isolation and purification of compounds useful in the present invention.
  • polypeptide' relates to proteins (such as TARGETS), proteinaceous molecules, fragments of proteins, monomers or portions of polymeric proteins, peptides, oligopeptides and enzymes (such as kinases, proteases, GPCR's etc.).
  • polynucleotide means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more particularly 70 percent of its base pairs are in common, particularly 80 percent, most particularly 90 per cent, and in a special embodiment 100 percent of its base pairs.
  • the polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof.
  • nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2'-0-(2- methoxy)ethylphosphorothioate.
  • PNA peptide nucleic acid
  • polysiloxane polysiloxane
  • 2'-0-(2- methoxy)ethylphosphorothioate The polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, particularly about 100 to about 4000 bases, more particularly about 250 to about 2500 bases.
  • One polynucleotide embodiment comprises from about 10 to about 30 bases in length.
  • polynucleotide is the polyribonucleotide of from about 17 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs - double stranded siRNA molecules or self-complementary single-stranded siRNA molecules (shRNA)).
  • small interfering RNAs siRNAs - double stranded siRNA molecules or self-complementary single-stranded siRNA molecules (shRNA)
  • nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2'-0-(2-methoxy)ethylphosphorothioate, or including non-naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.
  • Polynucleotides herein are selected to be 'substantially' complementary to different strands of a particular target DNA sequence.
  • the polynucleotides must be sufficiently complementary to hybridize with their respective strands. Therefore, the polynucleotide sequence need not reflect the exact sequence of the target sequence.
  • a non-complementary nucleotide fragment may be attached to the 5' end of the polynucleotide, with the remainder of the polynucleotide sequence being complementary to the strand.
  • non-complementary bases or longer sequences can be interspersed into the polynucleotide, provided that the polynucleotide sequence has sufficient complementarity with the sequence of the strand to hybridize therewith under stringent conditions or to form the template for the synthesis of an extension product.
  • the term 'preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
  • the term 'prophylaxis' is related to and encompassed in the term 'prevention', and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease.
  • prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
  • the term 'subject' includes humans and other mammals.
  • the term 'TARGET' or 'TARGETS' means the protein(s) identified in accordance with the assays described herein and determined to be involved in the down-regulation of a T h 2 cytokine phenotype.
  • the term TARGET or TARGETS includes and contemplates alternative species forms, isoforms, and variants, such as splice variants, allelic variants, alternate in frame exons, and alternative or premature termination or start sites, including known or recognized isoforms or variants thereof such as indicated in Table 1.
  • 'Therapeutically effective amount' or 'effective amount' means that amount of a compound or agent that will elicit the biological or medical response of a subject that is being sought by or is accepted by a medical doctor or other clinician.
  • the term 'treating' means an intervention performed with the intention altering the pathology of, and thereby ameliorating a disorder, disease or condition, including one or more symptoms of such disorder or condition.
  • the related term 'treatment,' as used herein, refers to the act of treating a disorder, symptom, disease or condition, as the term 'treating' is defined above.
  • the term 'treating' or 'treatment' of any disease or disorder refers to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof).
  • 'treating' or 'tretment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject.
  • 'treating' or 'treatment' refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter or of a physiologically measurable parameter), or both.
  • 'treating' or 'treatment' relates to slowing the progression of the disease.
  • vectors also relates to plasmids as well as to viral vectors, such as recombinant viruses, or the nucleic acid encoding the recombinant virus.
  • vertebrate cells means cells derived from animals having vertebral structure, including fish, avian, reptilian, amphibian, marsupial, and mammalian species. Preferred cells are derived from mammalian species, and most preferred cells are human cells. Mammalian cells include feline, canine, bovine, equine, caprine, ovine, porcine, murine, such as mice and rats, and rabbits.
  • T h 2 response is used throughout to refer to the processes involved in onset and transformation of T cells into T helper 2 type (T h 2 type). Many pathways and signaling molecules are involved in such response and are covered by this term.
  • T h l/T h 2 response imbalance refers to the imbalance between T h l and T h 2 cytokine response and consequently differentiation of na ' ive T cells in a T h l or T h 2 phenotype and subsequent accumulation of T h 2 phenotype.
  • biological responses might be a release of an inflammatory cytokines.
  • cytokines that activate T h 2 response include TSLP and GM-CSF.
  • T h 2 phenotype refers to the phenotypic properties of the T h 2 response expressed in elevated release and /or expression of signaling molecules, in particular cytokines.
  • T h 2 cytokine phenotype refers to the cytokine expression and/or release profile associated with the activation of T h 2 responses in cells.
  • the release and /or expression of signaling molecules, in particular cytokines are exemplary of, in each instance, a biological property related to the T h 2 phenotype.
  • T h 2-associated inflammatory disorder refers to the inflammatory disorders or conditions which are mainly and predominantly triggered by T h 2 response. Examples of such disorders include allergic conditions as well as inflammatory disorders like colitis, Crohn's disease, inflammatory bowel disease.
  • T h 2-associated fibrotic disorders include, but are not limited to, scleroderma, interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), liver fibrosis resulting from chronic hepatitis B or C infection, radiation- induced fibrosis, and fibrosis arising from wound healing
  • Applicant's invention is relevant to the treatment, prevention and alleviation of inflammatory responses and the treatment or prevention of inflammatory disorders or conditions.
  • the present invention provides methods for assaying for drug candidate compounds that down- regulate T h 2 response, comprising contacting the compound with a cell expressing a TARGET, and determining the relative amount or degree of T h 2-related responses produced by said cell in the presence and/or absence of the compound. Such methods may be used to identify target proteins that act to down- regulate T h 2 response; alternatively they may be used to identify compounds that down-regulate the expression or activity of TARGET proteins.
  • the invention provides methods for assaying for drug candidate compounds that down-regulate T h 2 response, comprising contacting the compound with a TARGET, under conditions wherein the activity of the TARGET may be measured, and determining whether the TARGET activity is altered in the presence of the compound.
  • the compound is contacted with a cell expressing the TARGET, and the relative amount or degree of T h 2-related responses produced by said cell in the presence and/or absence of the compound is determined.
  • Exemplary such methods can be designed and determined by the skilled artisan. Particular such exemplary methods are provided herein.
  • the present invention is based on the inventors' discovery that the TARGET polypeptides and their encoding nucleic acids, identified as a result of screens described below in the Examples, are factors involved in the regulation of TSLP release/production and therefore factors which are involved in the regulation of T h 2 inflammatory response.
  • a reduced activity or expression of the TARGET polypeptides and/or their encoding polynucleotides is causative, correlative or associated with reduced TSLP release by the cells.
  • a reduced activity or expression of the TARGET polypeptides and/or their encoding polynucleotides is causative, correlative or associated with reduced T h 2 response in cells.
  • the TARGET polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36-70 as listed in Table 1. Table 1
  • NM_001165979.1 23 NP_001159451.1 58 epsilon 1
  • PRKAR1A NM_002734.3 24 NP_002725.1 59 protein kinase, Kinase
  • NM_212471.1 25 NP 997636.1 60 cAMP-dependent, NM_212472.1 26 NP_997637.1 61 regulatory, type I,
  • PROCA1 NM_152465.1 28 NP_689678.1 63 protein interacting Phospholipase with cyclin Al
  • a particular embodiment of the invention comprises the protease TARGETS identified as SEQ ID NO: 36, 62, or 70.
  • a particular embodiment of the invention comprises the phospholipase TARGET identified as SEQ ID NO: 57-58 and 63
  • a particular embodiment of the invention comprises the ion channel TARGETS identified as SEQ ID NO: 45-48, and 65-69.
  • a particular embodiment of the invention comprises the kinase TARGETS identified as SEQ ID NO: 51-52, 56, 59-61 and 64.
  • a further particular embodiment of the invention comprises the GPCR TARGETS identified as SEQ ID NO: 49, 50, 53 and 55.
  • a particular embodiment of the invention comprises the hydrolase TARGETS identified as SEQ ID NOs: 42-44.
  • a particular embodiment of the invention comprises the oxidoreductase TARGETS identified as SEQ ID NO: 38-41 and 54.
  • a further particular embodiment of the invention comprises the extracellular TARGET identified as SEQ ID NO
  • the present invention relates to a method for assaying for drug candidate compounds that down-regulate T h 2 response, comprising contacting the compound with a polypeptide comprising an amino acid sequence of SEQ ID NO: 36-70, or a fragment or derivative thereof, under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound.
  • a polypeptide comprising an amino acid sequence of SEQ ID NO: 36-70, or a fragment or derivative thereof, under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound.
  • One particular means of measuring the complex formation is to determine the binding affinity of said compound to said polypeptide.
  • the present invention relates to a method for identifying a compound that down-regulates T h 2 response, said method comprising:
  • the present invention further relates to a method for identifying a compound that down-regulates T h 2 response, comprising:
  • the assay method might also be supplemented by additional steps of:
  • the invention relates to a method for identifying an agent that down-regulates T h 2 response, said method comprising:
  • the present invention relates to a method for assaying for drug candidate compounds that down-regulate T h 2 response, comprising contacting a compound with a polypeptide comprising an amino acid sequence of SEQ ID NO: 36-70, or a fragment thereof, under conditions that allow said compound to down-regulate the activity or expression of the polypeptide, and determining the activity or expression of the polypeptide.
  • One particular means of measuring the activity or expression of the polypeptide is to determine the amount of said polypeptide using a polypeptide binding agent, such as an antibody, or to determine the activity of said polypeptide in a biological or biochemical measure, for instance the amount of phosphorylation of a target of a kinase polypeptide.
  • a further means of measuring the activity or expression of the polypeptide is to determine the amount or extent of cytokines released by cell treated with the test compound.
  • such means is the measurement of the expression or release of inflammatory cytokines capable of inducing T h 2 response.
  • said measures of the activity of the TARGET polypeptide is the release or expression of TSLP or GM-CSF polypeptides.
  • the means for measurement of such polypeptides is known to a skilled artisan and many commercially available kits are available for detection and measurement of such proteins, additional specific methods are also described in the Examples herein.
  • a compound is identified as down-regulating the T h 2 response by measuring the effect on the T h 2 response, where a decrease in the markers of the T h 2 response is indicative of the down- regulation of the T h 2 response.
  • the ability of the compound to down-regulate T h 2 response can be measured by methods well known to those of skill in the art, including (without limitation) measuring the release of different cytokines or measuring expression of one or more cytokines or other signaling molecules known to induce T h 2 response which are recognized indicators of T h 2 response. Any signaling molecule known to a skilled artisan to be a potential inducer or indicator of T h 2 response might be used in the assay method.
  • cytokines selected from CXCL10, CXCL8, CCL5, CXCL5, CXCL2, CXCL3, CCL20, CXCL1 , CSF2, ⁇ , TSLP and GM-CSF are measured.
  • the expression or altered levels of TSLP or GM-CSF is measured. Any combination of the measurements might be performed in the same assay or sequentially in separate assays. The selection of a particular signaling molecule depends on the availability of reliable assays to measure such or the particular setup of the assay method.
  • MSD Meso Scale Discovery platform
  • MSD technology uses micro-plates with carbon electrodes integrated at the bottom of the plates; Biological reagents, immobilized to the carbon simply by passive adsorption, retain high biological activity.
  • MSD assays use electro-chemiluminescent labels (MSD-TAGTMlabel) for ultra-sensitive detection.
  • the detection process is initiated at electrodes located at the bottom of the micro-plates. Labels near the electrode only are excited and detected reducing background signal.
  • the antibodies for such assay might be purchased from different producers and the skilled artisan is in the position to choose correct antibodies to perform the assay.
  • the expression levels of the signaling molecules can be measured using known methods including quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR).
  • qPCR is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule.
  • Real Time-PCR enables both detection and quantification.
  • the quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.
  • the test compounds identified in one of the assay methods described above can be further selected based on whether the test compound affects IFN levels. Such additional criteria help to select a compound that can be used for the treatment of inflammatory disorders.
  • the candidate compound does not decrease IFN production and/or release by the cell.
  • the candidate compound is selected if it increases IFN production and/or release by the cell.
  • the assay method is performed as a cellular assay.
  • the requirement for such cell is that they express and/or produce the relevant signaling molecules capable of inducing or indicating T h 2 response.
  • such cells are epithelial cells derived from, preferably, a human subject.
  • said cellular assay is performed using are bronchial epithelial cell, preferably from a human subject. The choice of source of such is however not considered crucial for the invention method to be successfully performed. Alternatively, any mammalian cells naturally expressing TSLP can be utilized.
  • the cells are stimulated with one or more immunostimulants.
  • immunostimulants are known to a skilled artisan, however it might be preferred to use stimulants that provide a good read out for the method provided herein.
  • such stimulant is polyinosinic:polycytidylic acid (Poly I:C) with or without an addition of interleukin-4 (IL4).
  • Poly I:C is commonly used to simulate viral infections.
  • a virus for example but not by limitation a rhinovirus, might be used instead of Poly I:C.
  • the selection of an appropriate virus, e.g. rhinovirus serotype depends on the desired assay conditions and the chosen read-out.
  • the assay method uses a drug candidate compound identified as having a binding affinity for a TARGET, and/or has already been identified as having down- regulating activity such as antagonist activity vis-a-vis one or more TARGET.
  • Candidate compound or agents might be validated or rescreened in the TSLP -based assay described herein.
  • the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the polypeptide to thereby down-regulate T h 2 response.
  • an assay designed to determine the binding affinity of a compound to the polypeptide, or fragment thereof may be necessary, but may be one exemplary assay or one assay among additional and more particular or specific assays to ascertain whether the test compound would be useful for down-regulating T h 2 response, including particularly the T h 2 cytokine phenotype, when administered to a subject.
  • Suitable controls should always be in place to insure against false positive readings.
  • the screening method comprises the additional step of comparing the compound to a suitable control.
  • the control may be a cell or a sample that has not been in contact with the test compound.
  • the control may be a cell that does not express the TARGET; for example in one aspect of such an embodiment the test cell may naturally express the TARGET and the control cell may have been contacted with an agent, e.g. an siRNA, which inhibits or prevents expression of the TARGET.
  • the cell in its native state does not express the TARGET and the test cell has been engineered so as to express the TARGET, so that in this embodiment, the control could be the untransformed native cell.
  • the control may also alternatively utilize a known inhibitor of T h 2 response or a compound known not to have any significant effect on the T h 2 response. Whilst exemplary controls are described herein, this should not be taken as limiting; it is within the scope of a person of skill in the art to select appropriate controls for the experimental conditions being used.
  • the expression or release of TSLP or GM-CSF might be used as a property to assess if a compound down-regulates T h 2 response.
  • the levels of those proteins might be compared to a control.
  • Particular examples of such controls might include cells not treated with the compound for establishing the background level of TSLP or GM-CSF produced by the cells.
  • Other types of controls might include compounds known to have an effect on the expression or activity of the TARGET or the use of cells which do not express the TARGET.
  • the relative measure of the TSLP levels or expression, or of GM-CSF levels or expression, or such other T h 2 indicator may be taken.
  • T h 2 response such compound exhibit at least an effect of 40% decrease in TSLP or GM-CSF, etc. production compared to a control, in particular at least 50%, more particularly at least 60%.
  • the order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the compounds' binding affinity for the polypeptide. Alternatively, one may screen a set of compounds identified as having binding affinity for a polypeptide domain, or a class of compounds identified as being an inhibitor of the polypeptide. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds, a measurement of down-regulation of T h 2 response or of a biological property related to the T h 2 response is preferred.
  • T h 2 response The means by which to measure, assess, or determine the effect on T h 2 response, may be selected or determined by a skilled artisan. Validation studies including controls and measurements of binding affinity to the polypeptides or down-regulation of activity or expression of the polypeptides of the invention are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application.
  • the present assay method may be practiced in vitro, using one or more of the TARGET proteins, or fragments thereof, including monomers, portions or subunits of polymeric proteins, peptides, oligopeptides and enzymatically active portions thereof.
  • the binding affinity of a compound with the polypeptide TARGET can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore ® ), by saturation binding analysis with a labeled compound (for example, Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR ® ) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer.
  • the binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC 5 o or EC 5 o.
  • the IC 5 o represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide.
  • the EC 50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures TARGET function.
  • the dissociation constant, Kd is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide.
  • Compounds with a high affinity binding have low Kd, IC 50 and EC 50 values, for example, in the range of 100 tiM to 1 pM; a moderate- to low- affinity binding relates to high Kd, IC 50 and EC50 values, for example in the micromolar range.
  • polypeptides and polynucleotides useful in the practice of the present invention described herein may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • immobilize either the TARGET polypeptide or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay.
  • Interaction (for example, binding of) of the TARGET polypeptide with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix.
  • the TARGET polypeptide can be "His" tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the TARGET polypeptides can be adsorbed to IgG, which are then combined with the cell lysates (for example, (35)S-labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (for example, at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized.
  • the amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the TARGET protein quantified from the gel using standard electrophoretic techniques.
  • TARGET or the compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated TARGET protein molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (for example, biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with the TARGETS but which do not interfere with binding of the TARGET to the compound can be derivatized to the wells of the plate, and the TARGET can be trapped in the wells by antibody conjugation.
  • preparations of a labeled candidate compound are incubated in the wells of the plate presenting the TARGETS, and the amount of complex trapped in the well can be quantitated.
  • binding of a test compound to the TARGET can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates and test tubes.
  • a fusion protein can be provided which adds a domain that allows the TARGET polypeptide to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for concentration of salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly.
  • glutathione sepharose beads Sigma Chemical; St. Louis, Mo.
  • glutathione derivatized microtitre plates which are then combined with the test compound and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for concentration of salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly.
  • the present assay method may also be practiced in a cellular assay.
  • a host cell expressing the TARGET, or fragment(s) thereof can be a cell with endogenous expression or a cell modified to express or over-expressing the TARGET, for example, by transduction.
  • the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use host cells that over-express TARGET.
  • Over-expression has the advantage that the level of the TARGET substrate end-products is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier.
  • a non- endogenous form of TARGET may be expressed or over-expressed in a cell and utilized in screening.
  • the assay method may be based on the particular expression or activity of the TARGET polypeptide, including but not limited to an enzyme activity.
  • assays for the enzyme TARGETS identified as SEQ ID NOs: 36, 38-44, 51-52, 54, 56, 57-64, or 70 may be based on enzymatic activity or enzyme expression.
  • assays for the GPCR TARGET identified as SEQ ID NO: 49, 50, 53, or 55 may be based on GPCR activity or expression, including downstream mediators or activators.
  • Assays for ion channel TARGET identified as SEQ ID NO: 45-48, or 65-69 may use techniques well known to those of skill in the art including classical patch clamping, high-throughput fluorescence based or tracer based assays which measure the ability of a compound to open or close an ion channel thereby changing the concentration of fluorescent dyes or tracers across a membrane or within a cell.
  • the measurable phenomenon, activity or property may be selected or chosen by the skilled artisan.
  • the person of ordinary skill in the art may select from any of a number of assay formats, systems or design one using his knowledge and expertise in the art.
  • the present inventors have identified certain target proteins and their encoding nucleic acids by screening recombinant adenoviruses mediating the expression of a library of shRNAs, referred to herein as 'Ad-siRNAs'.
  • This type of library is a screen in which siRNA molecules are transduced into cells by recombinant adenoviruses, which siRNA molecules inhibit or repress the expression of a specific gene as well as expression and activity of the corresponding gene product in a cell.
  • Each siRNA in a viral vector corresponds to a specific natural gene.
  • a direct correlation can be drawn between the specific gene expression and a pathway involved in T h 2 responses.
  • the assay uses the measurement of the levels of TSLP or GM-CSF as known activators of T h 2 response.
  • the TARGET genes identified using the knock-down library are then used in the present inventive method for identifying compounds that can be used to in the treatment of diseases associated with the elevated T h 2 response, in particular of diseases where such elevated T h 2 response leads to imbalance in T h l/T h 2 responses.
  • the knock down (KD) target sequences identified in the Ad-siRNA screens more particularly described herein, include those set out below in the examples with SEQ ID NOs: 86-123) and shRNA compounds comprising those sequences have been shown herein to inhibit the expression and/or activity of these TARGET genes and the examples herein confirm the role of the TARGETS in the T h 2 response pathways.
  • Table 1 lists the TARGETS identified using applicants' knock-down library in the TSLP- based assay exemplified herein, including the class of polypeptides identified.
  • TARGETS have been identified in polypeptide classes including kinases, proteases, enzymes, ion channels, GPCRs, and extracellular proteins, for instance.
  • Ion channels are membrane protein complexes and their function is to facilitate the diffusion of ions across biological membranes.
  • Membranes, or phospholipid bilayers build a hydrophobic, low dielectric barrier to hydrophilic and charged molecules.
  • Ion channels provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane.
  • the activity of an ion channel can be measured using classical patch clamping.
  • High-throughput fluorescence-based or tracer-based assays are also widely available to measure ion channel activity. These fluorescent-based assays screen compounds on the basis of their ability to either open or close an ion channel thereby changing the concentration of specific fluorescent dyes across a membrane.
  • the tracer-based assay the changes in concentration of the tracer within and outside the cell are measured by radioactivity measurement or gas absorption spectrometry.
  • substrates are used in which a fluorescent group is linked to a quencher through a peptide sequence that is a substrate that can be cleaved by the target protease. Cleavage of the linker separates the fluorescent group and quencher, giving rise to an increase in fluorescence.
  • G-protein coupled receptors are capable of activating an effector protein, resulting in changes in second messenger levels in the cell.
  • the TARGETS represented by SEQ ID NO: 49, 50, 53, and 55 are GPCRs.
  • the activity of a GPCR can be measured by measuring the activity level of such second messengers.
  • Two important and useful second messengers in the cell are cyclic AMP (cAMP) and Ca 2+ .
  • cAMP cyclic AMP
  • Ca 2+ The activity levels can be measured by methods known to persons skilled in the art, either directly by ELISA or radioactive technologies or by using substrates that generate a fluorescent or luminescent signal when contacted with Ca 2+ or indirectly by reporter gene analysis.
  • the activity level of the one or more secondary messengers may typically be determined with a reporter gene controlled by a promoter, wherein the promoter is responsive to the second messenger.
  • Promoters known and used in the art for such purposes are the cyclic- AMP responsive promoter that is responsive for the cyclic- AMP levels in the cell, and the NF-AT responsive promoter that is sensitive to cytoplasmic Ca 2+ -levels in the cell.
  • the reporter gene typically has a gene product that is easily detectable.
  • the reporter gene can either be stably infected or transiently transfected in the host cell.
  • Useful reporter genes are alkaline phosphatase, enhanced green fluorescent protein, destabilized green fluorescent protein, luciferase and ⁇ -galactosidase.
  • the cells expressing the polypeptides may be cells naturally expressing the polypeptides, or the cells may be may be transfected to express the polypeptides, as described above. Also, the cells may be transduced to overexpress the polypeptide, or may be transfected to express a non- endogenous form of the polypeptide, which can be differentially assayed or assessed.
  • the methods of the present invention further comprise the step of contacting the population of cells with an agonist of the polypeptide. This is useful in methods wherein the expression of the polypeptide in a certain chosen population of cells is too low for a proper detection of its activity. By using an agonist the polypeptide may be triggered, enabling a proper read-out if the compound inhibits the polypeptide.
  • the population of cells may be exposed to the compound or the mixture of compounds through different means, for instance by direct incubation in the medium, or by nucleic acid transfer into the cells.
  • transfer may be achieved by a wide variety of means, for instance by direct transfection of naked isolated DNA, or RNA, or by means of delivery systems, such as recombinant vectors.
  • Other delivery means such as liposomes, or other lipid-based vectors may also be used.
  • the nucleic acid compound is delivered by means of a (recombinant) vector such as a recombinant virus.
  • libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (for example, LOPAPTM, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (for example, LOPACTM, Sigma Aldrich, BioFocus) or natural compound libraries (Specs, TimTec, BioFocus).
  • Particular drug candidate compounds are low molecular weight compounds.
  • Low molecular weight compounds for example with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et ah, 2001)).
  • Peptides comprise another particular class of drug candidate compounds. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors.
  • Natural compounds are another particular class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized. The lipids are another particular class of drug candidate compound.
  • Another particular class of drug candidate compounds is an antibody.
  • the present invention also provides antibodies directed against a TARGET. These antibodies may be endogenously produced to bind to the TARGET within the cell, or added to the tissue to bind to TARGET polypeptide present outside the cell. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of a Fab expression library, and Fv fragments and the products of an Fv expression library.
  • the compound may be a nanobody, the smallest functional fragment of naturally occurring single-domain antibodies (Cortez-Retamozo et al. 2004).
  • the antibodies include a domain antibody (dAb) fragment, which comprises a single variable domain; a camelid antibody; an isolated complementarity determining region (CDR); a Single Chain Fv Fragment; a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; and other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.
  • dAb domain antibody
  • CDR isolated complementarity determining region
  • Single Chain Fv Fragment a diabody, which
  • polyclonal antibodies may be used in the practice of the invention.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • Antibodies may also be generated against the intact TARGET protein or polypeptide, or against a fragment, derivatives including conjugates, or other epitope of the TARGET protein or polypeptide, such as the TARGET embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library.
  • the immunizing agent may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate
  • the antibodies may be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using methods known in the art.
  • the monoclonal antibodies of the present invention may be "humanized” to prevent the host from mounting an immune response to the antibodies.
  • a "humanized antibody” is one in which the complementarity determining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins.
  • Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa.
  • the humanization of antibodies may be accomplished by methods known in the art (see, for example, Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer- Verlag, New York). Transgenic animals may be used to express humanized antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol. 222:581-97). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(l):86-95).
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.
  • Bispecific antibodies are monoclonal, particularly human or humanized, antibodies that have binding specificities for at least two different antigens and particularly for a cell-surface protein or receptor or receptor subunit.
  • one of the binding specificities is for one domain of the TARGET, while the other one is for another domain of the same or different TARGET.
  • bispecific antibodies Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule. Similar procedures are disclosed in Trauneeker, et al. (1991) EMBO J. 10:3655-9.
  • the present invention provides a method for identifying a compound that down-regulates
  • T h 2 response said method comprising:
  • the method might be performed in vitro in a cell-free preparation.
  • the binding of a compound to the nucleic acid might be established in a cellular assay.
  • such assay can be performed using mammalian cells, in particular epithelial cells.
  • such nucleic acid is a RNA sequence.
  • RNA sequence might be a result of transcription of TARGET sequences or might be artificially synthesized.
  • the identified compound is an inhibitor of T h 2 response.
  • the compounds that are tested for binding to the TARGET polynucleotide might be preselected based on their potential of binding to the specific TARGET polynucleotide. However, not all compounds that bind to the TARGET will demonstrate the effect on down-regulation of T h 2 response and, therefore, a measurement of a biological property related to the down-regulation of T h 2 phenotype might be desired.
  • a TARGET polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the TARGET polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to a control. Selection of appropriate controls in known to a skilled artisan. In particular the effect of the test compound might be compared to a level of mRNA expression in the absence of the compound. Alternatively possible controls might be compound that are known to significantly down- regulate the expression of mRNA and compound which are known to have no effect of the expression of TARGET mRNA. The test compound can then be identified as a regulator of expression based on such comparisons.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • TARGET gene expression can be measured using techniques well-known to a skilled artisan. Particular examples of such techniques include northern analysis or real-time PCR. Those methods are indicative of the presence of nucleic acids encoding TARGETS in a sample, and thereby correlates with expression of the transcript from the polynucleotide.
  • a compound is identified as down-regulating the T h 2 response by measuring the effect on the T h 2 response, where a decrease of the markers of the T h 2 response is indicative of the down-regulation of the T h 2 response. In a specific aspect the compound inhibits T h 2 response.
  • the ability of the compound to down-regulate T h 2 response might be measured by methods well known to those of skill in the art, including (without limitation) using different cytokine level measurements or measurements of expression of one or more cytokines or other signaling molecules known to induce or indicate T h 2 response. Any signaling molecule known to a skilled artisan to be a potential inducer of T h 2 response might be used in the assay method.
  • the expression or release of one or more cytokines selected from CXCL10, CXCL8, CCL5, CXCL5, CXCL2, CXCL3, CCL20, CXCL1, CSF2, ⁇ , TSLP or GM-CSF are measured.
  • the level of TSLP or GM-CSF is measured. Any combination of the measurements might be performed in the same assay or sequentially in separate assays. The selection of a particular signaling molecule depends on the availability of reliable assays to measure such or the particular setup of the assay method.
  • test compounds identified in one of the assay methods described above can be further selected based on whether the test compound affects IFN levels.
  • additional criteria help to select a compound that can be used for the treatment of inflammatory disorders.
  • IFN has been reported to show an antiviral activity which is mainly involved in innate immune response.
  • the candidate compound does not decrease IFN production and/or release by the cell.
  • the candidate compound is selected if it increases IFN production and/or release by the cell.
  • the assay method is performed as a cellular assay.
  • the requirement for such cell is that they express and/or produce the relevant signaling molecules capable of inducing or indicating T h 2 response.
  • such cells are epithelial cells derived from, preferably, a human subject.
  • said cellular assay is performed using are bronchial epithelial cell, preferably from a human subject. The choice of source of such is however not considered crucial for the invention method to be successfully performed. Alternatively, any mammalian cells naturally expressing TSLP can be utilized.
  • the cells are stimulated with one or more immunostimulants.
  • Such immunostimulants are known to a skilled artisan, however it might be preferred to use stimulants that provide a good read out for the method provided herein.
  • such stimulant is polyinosinic:polycytidylic acid (Poly I:C) with or without an addition of interleukin-4 (IL4).
  • Poly I:C is commonly used to simulate viral infections.
  • a virus for example but not by limitation a rhinovirus, might be used instead of Poly I:C.
  • the selection of an appropriate virus e.g.
  • the present invention provides a method for identifying a compound that down-regulates T h 2 response wherein said compound is selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally- occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a said polynucleotide.
  • siRNA small interfering RNA
  • the present invention further relates to a method for identifying a compound that down- regulates T h 2 response comprising contacting mammalian cells with said compound comprising a polyribonucleotide sequence that complements at least about 15 to 30, particularly at least 17 to 30, most particularly at least 17 to 25, more specifically at least 19 to 21 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1-35.
  • such compounds are identified as expression inhibitory agents.
  • Another aspect of the present invention relates to a method for identifying a compound that down-regulates the Th2 response, comprising contacting mammalian cells with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding a TARGET polypeptide.
  • a particular embodiment relates to a composition comprising a polynucleotide including at least one antisense strand that functions to pair the agent with the TARGET mRNA, and thereby down-regulate or block the expression of TARGET polypeptide.
  • the inhibitory agent particularly comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally- occurring polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-35.
  • siRNA small interfering RNA
  • a special embodiment of the present invention relates to a method for identifying a compound that down-regulates T h 2 response
  • the compound is an expression-inhibiting agent and is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 36-70, a small interfering RNA (siRNA, particularly shRNA) that is sufficiently homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 1-35, such that the antisense RNA, ODN, ribozyme, particularly siRNA, particularly shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
  • antisense RNA antisense oligodeoxynucleotide
  • ODN antisense oligodeoxynucleotide
  • shRNA small interfer
  • the TARGET is a phospholipase
  • the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 57-58 or 63 or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 22-23 or 28, exemplary oligonucleotide sequences include SEQ ID NO: 96, 99, 116, 119, 135, 154, 156, and 157.
  • the TARGET is an ion channel, therefore the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 45-48, 65-68, or 69 or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 10-13, 30-33, or 34, exemplary oligonucleotide sequences include SEQ ID NO: 88, 101, 102, 108, 121, and 122 .
  • the TARGET is a kinase, therefore the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 51-52, 56, 59-61, or 64, or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 16-17, 21, 24-26, or 29, exemplary oligonucleotide sequences include SEQ ID NO: 91, 95, 97, 100, 111, 115, 117, 120, 134, 136, 138, 150, and 153.
  • the TARGET is a protease
  • the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 36, 62, or 70 or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 1, 27, or 35
  • exemplary oligonucleotide sequences include SEQ ID NO: 84, 98, 103, 104, 118, 123, 124, 137, 142, 155, and 159.
  • the TARGET is a GPCR
  • the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 49, 50, 53, or 55 or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 14, 15, 18, or 20
  • exemplary oligonucleotide sequences include SEQ ID NO: 89, 90, 92, 94, 119, 110, 112, 114, 129, 130, 131, 133, 147, 148,149, and 151.
  • the TARGET is a hydrolase
  • the ribozyme may cleave a polynucleotide coding for SEQ ID NO: 42-44 or the siRNA or shRNA is homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 7-9
  • exemplary oligonucleotide sequences include SEQ ID NO: 87, 107, 127 and 145. Further exemplary oligionucleotide sequences useful for practicing the invention can be found in the Examples. These examples, however, should not be considered limiting and are only provided to illustrate the invention.
  • Another embodiment of the present invention relates to a method for identifying a compound that down-regulates T h 2 response
  • said compound is an expression-inhibiting agent such as a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide corresponding to SEQ ID NO: 1-35, a small interfering RNA (siRNA, particularly shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide corresponding to SEQ ID NO: 1-35 , such that the antisense RNA, ODN, ribozyme, particularly siRNA, particularly shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
  • an expression-inhibiting agent such as a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a
  • the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, particularly shRNA, comprising a polyribonucleotide sequence that complements at least about 17 to about 30 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-35
  • the expression- inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, particularly shRNA, comprising a polyribonucleotide sequence that complements at least 15 to about 30, particularly at least 17 to about 30, most particularly at least 17 to about 25, more specifically at least 19 to about 21 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-35.
  • a special embodiment comprises a polyribonucleotide sequence that complements a polynucleotide sequence selected from the group
  • Antisense nucleic acids of the invention are particularly nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a TARGET polypeptide or the corresponding messenger RNA.
  • antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a TARGET polypeptide by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for a TARGET.
  • the antisense sequence is at least about 15-30, and particularly at least 17 nucleotides in length.
  • the preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art.
  • One embodiment of compound is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-35.
  • an antisense nucleic acid for example, DNA
  • Antisense oligonucleotides may comprise a sequence containing from about 15 to about 100 nucleotides, more particularly from 15 to 30 nucleotides, and most particularly, from about 17 to about 25 nucleotides.
  • Antisense nucleic acids may be prepared from about 15 to about 30 contiguous nucleotides selected from the sequences of SEQ ID NO: 1-35, expressed in the opposite orientation. The binding of such nucleic acids to the TARGET sequence can be tested in a cell- free preparation as well as in a cellular assay.
  • Antisense oligonucleotides may be selected on the basis of secondary structure (Wickstrom et al (1991) in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wickstrom, ed., Wiley-Liss, Inc., New York, pp. 7-24; Lima et al. (1992) Biochem. 31 :12055-12061). Schmidt and Thompson (U.S.
  • Patent 6,416,951 describe a method for identifying a functional antisense agent comprising hybridizing an RNA with an oligonucleotide and measuring in real time the kinetics of hybridization by hybridizing in the presence of an intercalation dye or incorporating a label and measuring the spectroscopic properties of the dye or the label's signal in the presence of unlabelled oligonucleotide.
  • any of a variety of computer programs may be utilized which predict suitable antisense oligonucleotide sequences or antisense targets utilizing various criteria recognized by the skilled artisan, including for example the absence of self- complementarity, the absence of hairpin loops, the absence of stable homodimer and duplex formation (stability being assessed by predicted energy in kcal/mol).
  • Examples of such computer programs are readily available and known to the skilled artisan and include the OLIGO 4 or OLIGO 6 program (Molecular Biology Insights, Inc., Cascade, CO) and the Oligo Tech program (Oligo Therapeutics Inc., Wilsonville, OR).
  • antisense oligonucleotides suitable in the present invention may be identified by screening an oligonucleotide library, or a library of nucleic acid molecules, under hybridization conditions and selecting for those which hybridize to the target RNA or nucleic acid (see for example U.S. Patent 6,500,615). Mishra and Toulme have also developed a selection procedure based on selective amplification of oligonucleotides that bind target (Mishra et al (1994) Life Sciences 317:977-982).
  • Oligonucleotides may also be selected by their ability to mediate cleavage of target RNA by RNAse H, by selection and characterization of the cleavage fragments (Ho et al (1996) Nucl Acids Res 24: 1901-1907; Ho et al (1998) Nature Biotechnology 16:59-630). Generation and targeting of oligonucleotides to GGGA motifs of RNA molecules has also been described (U.S. Patent 6,277,981).
  • the antisense nucleic acids are particularly oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both.
  • the antisense nucleic acids can be synthetic oligonucleotides.
  • the oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity.
  • Specific examples of some particular oligonucleotides envisioned for this invention include those containing modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage.
  • Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases.
  • the RNA-DNA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.
  • RNase ribonuclease
  • Oligonucleotides may also contain one or more substituted sugar moieties.
  • Particular oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation.
  • chemical groups can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its TARGET site. Modifications may include 2'-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art.
  • Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains.
  • the substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its TARGET sequence.
  • the catalytic portion cleaves the TARGET RNA at a specific site.
  • the substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence. The ribozyme recognizes and then binds a TARGET mRNA through complementary base pairing.
  • the ribozyme acts enzymatically to cut the TARGET mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its TARGET sequence, it is released and can repeatedly bind and cleave at other mRNAs.
  • Exemplary ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif.
  • Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9).
  • a ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21 :3249-55).
  • Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the TARGET mRNA after transcription.
  • the oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers.
  • the amplification product is cloned into a eukaryotic expression vector.
  • Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors.
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21 :2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).
  • a particular inhibitory agent is a small interfering RNA (siRNA, particularly small hairpin
  • siRNA particularly shRNA, mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA.
  • siRNA according to the present invention comprises a sense strand of 15-30, particularly 17-30, most particularly 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of sequences described in SEQ ID NO: 1-35, particularly from the group of sequences described in SEQ ID NOs:84-159, and an antisense strand of 15-30, particularly 17-30, most particularly 17-25, more specifically 19-21 nucleotides complementary to the sense strand.
  • the most particular siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the TARGET polynucleotide sequence.
  • the siRNA further comprises a loop region linking the sense and the antisense strand.
  • a self-complementing single stranded shRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker.
  • the loop region sequence is 4-30 nucleotides long, more particularly 5-15 nucleotides long and most particularly 8 or 12 nucleotides long.
  • the linker sequence is UUGCUAUA (SEQ ID NO: 71) or GUUUGCUAUAAC (SEQ ID NO: 72).
  • Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.
  • the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars and/or chemical linkage the siRNA to one or more moieties or conjugates.
  • the nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO 2004/094636 and US 2003/0198627, are hereby incorporated by reference).
  • the present invention also relates to methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of down-regulating a T h 2 response and described hereinabove as an expression inhibitory agent.
  • a special aspect of these methods relates to the down-regulation or blocking of the expression of a TARGET polypeptide by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the TARGET polypeptide.
  • An intracellular binding protein includes an activity-inhibitory agent and any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide.
  • the intracellular binding protein may be an antibody, particularly a neutralizing antibody, or a fragment of an antibody or neutralizing antibody having binding affinity to an epitope of the TARGET polypeptide of SEQ ID NO: 36-70. More particularly, the intracellular binding protein is a single chain antibody.
  • a special embodiment of these methods comprises the expression-inhibitory agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 36-70, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide corresponding to SEQ ID NO: 1-35, such that the siRNA interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
  • ODN antisense oligodeoxynucleotide
  • siRNA small interfering RNA
  • the polynucleotide expressing the expression-inhibiting agent, or a polynucleotide expressing the TARGET polypeptide in cells is particularly included within a vector.
  • the polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, particularly, recombinant vector constructs, which will express the nucleic acid or antisense nucleic acid once the vector is introduced into the cell.
  • a variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendai viral vector systems. All may be used to introduce and express polynucleotide sequence for the expression- inhibiting agents in TARGET cells.
  • the viral vectors used in the methods of the present invention are replication defective.
  • Such replication defective vectors will usually lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non- functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the replication defective virus retains the sequences of its genome, which are necessary for encapsidating the viral particles.
  • the viral element used in the method is derived from an adenovirus.
  • the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof.
  • Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a particular vehicle for incorporating in a library of the invention.
  • An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells.
  • the vehicle includes adenoviral fiber proteins from at least two adenoviruses.
  • Particular adenoviral fiber protein sequences are serotype 17, 45 and 51. Techniques or construction and expression of these chimeric vectors are disclosed in US 2003/0180258 and US 2004/0071660, hereby incorporated by reference.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof.
  • An adenoviral late protein for instance an adenoviral fiber protein, may be favorably used to TARGET the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell.
  • the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and/or analogue thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and/or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell.
  • the adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention.
  • Retroviral vector systems are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus” MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus. Lentiviral vector systems may also be used in the practice of the present invention. Retroviral systems and herpes virus system may be particular vehicles for transfection of neuronal cells.
  • MSV murine Moloney leukemia virus
  • HaSV Hardvey sarcoma virus
  • SNV spleen necrosis virus
  • RSV Ra sarcoma virus
  • Friend virus Friend virus.
  • Lentiviral vector systems may also be used in the practice of the present invention. Retroviral systems and herpes virus system may be particular vehicles for transfection of
  • adeno-associated viruses are utilized.
  • the AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site- specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc.
  • Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters.
  • the promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, T7, lambda P.sub.r, P.sub. l, and trp promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (for example, HPRT, vimentin, actin, tubulin), intermediate filament promoters (for example, desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (for example, MDR type, CFTR, factor VIII), tissue-specific promoters (for example, actin promoter in smooth muscle cells, or Fit and Flk promoters active in endothelial cells), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), albumin gene control region which is active in liver (Pinkert, et al. (1987) Genes and Devel. 1 :268-76), alpha-fetoprotein gene control region which is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1 -antitrypsin gene control region which is active in the liver (Kelsey, et al.
  • beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al. (1987) Cell 48:703- 12), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, (1985) Nature 314.283-6), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al. (1986) Science 234:1372-8).
  • promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (for example, steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate- early, retroviral LTR, metallothionein, SV-40, Ela, and MLP promoters.
  • Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient, for example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection.
  • Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31 ; Ulmer, et al. (1993) Science 259:1745-8).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127.
  • the use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides for example, hormones or neurotransmitters, and proteins, for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopeptide (for example, WO 95/21931), peptides derived from DNA binding proteins (for example, WO 96/25508), or a cationic polymer (for example, WO 95/21931).
  • DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, for example, Wilson, et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263 :14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl.
  • Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).
  • a biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the agent, compound, polynucleotide, polypeptide, vector, or antibody of the invention is maintained in an active form, for example, in a form able to affect a biological activity.
  • a compound of the invention would have inverse agonist or antagonist activity on the TARGET; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of a TARGET; a vector would be able to transfect a TARGET cell and express the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a TARGET polypeptide domain.
  • a particular biologically compatible composition is an aqueous solution that is buffered using, for example, Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels.
  • Biologically compatible solutions may include stabilizing agents and preservatives.
  • the biocompatible composition is a pharmaceutically acceptable composition.
  • Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques.
  • the composition may be administered parenterally in dosage unit formulations containing standard, well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired.
  • a particular embodiment of the present invention is a pharmaceutical composition for down-regulating the T h 2 response ,comprising a therapeutically effective amount of an agent selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally- occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-35 in admixture with a pharmaceutically acceptable carrier.
  • an agent selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA)
  • siRNA small interfering RNA
  • Another particular embodiment is a pharmaceutical composition for the treatment or prevention of a condition related to an elevated T h 2 response. More specific embodiment is a pharmaceutical composition for the treatment or prevention of conditions or diseases associated with T h l/T h 2 imbalance.
  • conditions and diseases include, but are not limited to, asthma, allergic rhinitis, atopic dermatitis, fibrosis, atherosclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, glomerulonephritis, cryoglobulinemia, immune deficiency, rhinoconjunctivitis, anaphylaxis, bladder cancer, lung cancer, breast cancer, melanoma, colon and rectal cancer, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer, leukemia, and thyroid cancer.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • Particular sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent.
  • pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (for example, monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di- glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
  • the compounds or compositions of the invention may be combined for administration with or embedded in polymeric carrier(s), biodegradable or biomimetic matrices or in a scaffold.
  • the carrier, matrix or scaffold may be of any material that will allow composition to be incorporated and expressed and will be compatible with the addition of cells or in the presence of cells. Particularly, the carrier matrix or scaffold is predominantly non-immunogenic and is biodegradable.
  • biodegradable materials include, but are not limited to, polyglycolic acid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suture material, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin, alginate, cotton, or other naturally- occurring biodegradable materials.
  • the matrix or scaffold material may be sterilized prior to administration or implantation, e.g., by treatment with ethylene oxide or by gamma irradiation or irradiation with an electron beam.
  • a number of other materials may be used to form the scaffold or framework structure, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox (TPX), polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and a variety of polyhydroxyalkanoates, and combinations thereof.
  • nylon polyamides
  • dacron polymers
  • polystyrene polyprop
  • Matrices suitable include a polymeric mesh or sponge and a polymeric hydrogel.
  • the matrix is biodegradable over a time period of less than a year, more particularly less than six months, most particularly over two to ten weeks.
  • the polymer composition, as well as method of manufacture, can be used to determine the rate of degradation. For example, mixing increasing amounts of polylactic acid with polyglycolic acid decreases the degradation time.
  • Meshes of polyglycolic acid that can be used can be obtained commercially, for instance, from surgical supply companies (e.g., Ethicon, N.J). In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
  • the composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available.
  • a hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.
  • Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the agent of the present invention and a transfection enhancer, such as poloxamer.
  • Poloxamer 407 An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.) and is a non-toxic, biocompatible polyol.
  • a poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
  • the active agents may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • Pharmaceutical compositions that exhibit large therapeutic indices are particular.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the dosage of such compounds lies particularly within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to targeted tissues, complexed with cationic lipids, packaged within liposomes, or delivered to targeted cells by other methods known in the art. Localized administration to the desired tissues may be done by direct injection, transdermal absorption, catheter, infusion pump or stent.
  • the DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment.
  • Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and administration are provided in Sullivan et al. WO 94/02595.
  • Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time.
  • Those skilled in the art may employ different formulations for polynucleotides than for proteins.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention.
  • Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 104 and about 1014 pfu. In the case of AAVs and adenoviruses, doses of from about 106 to about 1011 pfu are particularly used.
  • pfu plaque-forming unit
  • plaque-forming unit corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.
  • Administration of the expression-inhibiting agent of the present invention to the subject patient includes both self-administration and administration by another person.
  • the patient may be in need of treatment for an existing disease or medical condition, or may desire prophylactic treatment to prevent or reduce the risk for diseases and medical conditions affected by a disturbance in bone metabolism.
  • the expression-inhibiting agent of the present invention may be delivered to the subject patient orally, transdermally, via inhalation, injection, nasally, rectally or via a sustained release formulation.
  • the present invention further provides use of agents described above in the manufacture of a medicament for the treatment and/or prevention of conditions or diseases involving elevated T h 2 response.
  • the disease is selected from asthma, allergic rhinitis, atopic dermatitis, fibrosis, atherosclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, glomerulonephritis, cryoglobulinemia, immune deficiency, rhinoconjunctivitis, anaphylaxis, bladder cancer, lung cancer, breast cancer, melanoma, colon and rectal cancer, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer, leukemia, thyroid cancer.
  • the disease is asthma.
  • T h 2 cytokine phenotype is responsible for many conditions associated with imbalance in T h l/T h 2 response.
  • the following assays, when used in combination with arrayed adenoviral shRNA (small hairpin RNA) or adenoviral cDNA expression libraries (the production and use of which are described in W099/64582), or siRNA libraries compounds or compound libraries are useful for the discovery of factors that are capable of down-regulating the T h 2 inflammatory response.
  • Example 1 describes the set-up and optimization of the screening assay conditions using
  • Example 2 describes primary screen using over 11000 adenoviral shRNA constructs
  • Example 3 describes re-screen of the hit obtained in primary screen using independently- propagated shRNA constructs
  • Example 4 describes the "on-target” validation further used for validation of the hits
  • Example 5 describes secondary assay which may be used for further confirmation of the hits
  • Example 6 describes a methodology for elimination of false positive hits due to toxic effects in cellular screens
  • Example 7 describes the profiling the effect of the TARGET(s) knock-down by synthetic siRNA approach on innate IFNB1 responses by triggering with PolyLC.
  • Example 1. Design and optimization of the TSLP secretion assay
  • NHBE normal human bronchial epithelial
  • NHBE cells were obtained at passage 1 and were cultured in specified BEGM obtained from Lonza (cat num. CC-3170). Cells were expanded to a maximal confluency of -80% to avoid senescence and were stored as passage 2. To ensure equal cell conditions, NHBE cells are used at passage 3 for experiments during assay development, screening and validation. Cell growth of NHBE cells was tested in 96-well as well as 384-well format. Although NHBE cells did grow in 384-wells, well-to-well variation in cell numbers was observed, that was not observed in 96-well plates. To prevent biased results in TSLP measurements due to cell variation, the 96-well format was selected for further assay development and screening.
  • TSLP ELISAs are commercially available with a sensitivity of approximately 30 pg/mL, it was anticipated that these may not be sensitive enough to detect the expected low TSLP levels induced by the trigger rhinovirus RV16 (Kato, 2007). Therefore, a Meso Scale Discovery (MSD) prototype human TSLP assay was optimized. Two variants of the assay, differing in the detection antibody labeling, were tested. Initially, a pre-mix of biotinylated anti-TSLP and Streptavidin-SULFO- TAG (indirectly labeled detection antibody (Ab)) was used. Due to availability issues, the indirectly labeled Ab was replaced by a SULFO-TAG labeled anti-TSLP Ab (directly labeled Ab).
  • MSD Meso Scale Discovery
  • Both assay variants demonstrated a reproducible and robust standard curve with an estimated detection limit of 0.5-1 pg/mL TSLP in both 96-well as well as 384-well format.
  • the absolute signals obtained with the directly labeled detection Ab were approximately two-fold lower compared to those obtained with the indirectly labeling, this did not affect the sensitivity of the assay as the associated background levels were also lower.
  • Assessing the detection limit revealed a detection limit well below the lowest TSLP concentration (0.6 pg/mL) on the standard curve.
  • the optimized 384-well MSD TSLP assay appeared to be >30-fold more sensitive compared to standard TSLP ELISA's and hence was preferred as read-out parameter for assays.
  • the screening assay involved the measurement of secreted TSLP in supernatants of NHBE cells following triggering with Poly I:C alone or in combination with cytokines.
  • Cells from donors 1, 2 and 3 were treated with increasing doses of Poly I:C, IL4 or with a combination of both and TSLP secretion was measured using TSLP MSD assay (Optimization of assay set-up, revealed optimal conditions when seeding -4000 cells/well in 96 well plates, adding triggers after 6-7 days and measuring TSLP secretion 24- 48 h after trigger).
  • Activation of the NHBE cells with Poly I:C showed a dose- dependent induction of TSLP secretion in all three donors tested.
  • a selection of shRNA adenoviruses directed to either GFP or luciferase were tested as potential negative knockdown (KD) controls. These negative control viruses were tested on at different MOI using poly I:C + IL-4 as trigger. Only three out of ten control viruses affected the readout with more than 25%, and only one control virus (ffluc_v30) had a significant effect.
  • a set of four negative control viruses was selected (ffluc_vl9, aveGFP_vl3, luc_vl3 and ffluc_v22) that reproducibly showed ⁇ 25% inhibition or induction of TSLP secretion following activation with pI:C + IL-4 in three separate experiments. This initial set was further re-propagated and tested in assay development.
  • a set of ⁇ 300 shRNA C20 adenoviruses targeting 70 genes was generated that could function as potential positive knockdown (KD) controls.
  • the target genes of these putative positive control viruses were derived from literature and are genes in the pathways or closely related to the pathways involved in the response to either rhinovirus or Poly I:C.
  • a set of seven viruses gave more than 40% inhibition compared to negative control viruses and therefore this set of seven viruses was chosen for further testing in assay development.
  • the selected positive controls for further assay development were: IRAK2_v9, RELA_v2, SYK_v7, TICAMl_v5, TLR3_v8, TLR9_v9, and TSLP_v3 (sequences for the positive and negative controls listed in Table 2).
  • TLR3_v8 80 GAACTAAAGATCATCGATT
  • TLR9_v9 82 GCCATACCAACATCCTGAT
  • TLR3-induced signaling pathways are triggered by binding of double- stranded RNA, a molecular pattern associated with viral infection such as rhinovirus.
  • Poly I:C a synthetic analog of dsRNA, is specifically recognized by TLR3.
  • One pathway activated by the TLR- 3 is the NF-kappaB pathway (Jiang, 2004).
  • the NF-kappaB family of transcription factors is composed of five members— p65 (REL-A), REL-B, cytoplasmic (c) REL, p50 and p52— which function as homo- and heterodimers (Akira & Takeda, 2004). It was demonstrated that IRAK-2 has a central role in TLR signaling pathways to NF-kappaB activation (Keating, 2007). IRAK-2 seems to act upstream of the TRIF-dependent pathway as IRAK-2 can interact with TLR3. In addition, it appears that also Syk signaling is important for multiple TLR signaling events and functions (Manukyan, 2009).
  • an assay was set-up with robust and reproducible results using pI:C (1.56 ⁇ g/mL) and IL-4 (0.1 ng/nL) as trigger in donor 3 with a designated set of negative and positive controls. This assay was used to perform the screens.
  • a primary screen was performed using over 1 1 ,000 adenoviral shRNA constructs comprising the SilenceSelect® library.
  • the full screen consisted of 127 96-well plates and was performed in biological duplicate.
  • the assay set up for the primary screen is outlined in Figure 1. 1.56 ⁇ g/mL polyLC + 0.1 ng/mL IL-4 were used as trigger on NHBE cells from donor 3.
  • Each individual 96-well plate contained 3 negative (ffluc_vl9, aveGFP_vl3 and luc_vl3) and 5 positive (IRAK2_v9, RELA_v2, TLR3_v8, TICAMl_v5 and TSLP_v3) controls in a layout as indicated on Figure 2.
  • Primary screen plates were considered successful if biological duplicates showed a Spearman rank correlation of >0.4 or a Kappa value >0.2. The performance of the screen was considered of good quality, if upon hit calling at least 3 out of 5 positive controls anda maximum of one negative control (false positive) showed up as hits.
  • the re-screen was performed using similar test conditions as in Example 2 but in a different donor to eliminate donor specific effects of viruses.
  • the primary hit viruses were re-propagated and NHBE cells from donor 4 were seeded and transduced in a layout shown in Figure 4.
  • hit-calling in the rescreen would be based on negative controls, at least 30% of the wells in each plate consisted of negative controls while 5 positive controls were included in column 7.
  • Cells were then triggered with 1.56 ⁇ g/mL Poly I:C + 0.1 ng/mL IL-4 and TSLP secretion was measured 24 h later. Following a pilot rescreen to determine the optimal viral dilution for this donor, the complete rescreen was performed in one batch.
  • NPI normalized percentage inhibition method
  • RSA redundant siRNA activity
  • an assay was set up using NHBE cells from donor 7 - a third donor - that were transfected with 20 nM siRNA and subsequently triggered with 6.25 ⁇ g/mL polyLC + 5 ng/mL IL-4 according to the layout shown in Figure 5.
  • Synthetic siRNA purchased from Dharmacon (ONTARGETplus, smartpools) and Ambion
  • siRNAs targeting TIC AMI can efficiently inhibit TSLP secretion induced by 6.25 ⁇ g/mL Poly I:C + 5 ng/mL IL-4.
  • siRNAs for each of the confirmed targets were ordered. 3 different siRNAs were delivered and tested on cells using the protocol described above. To enable hit calling, each plate in the on-target screen contained 30% negative control siRNAs as indicated in Figure 6.
  • the on-target screen was done in biological duplicate and was done twice at separate occasions. Data were Log2 transformed and corrected for an observed plate edge effect using a model that was based on the data of all negative controls within each of the 2 runs. NPI was calculated based on the window between the average of the negative (0 %) and positive controls (100 %) on each plate and subjected to quality metrics using the Strictly Standardized Mean Difference (SSMD) method (Zhang 2008); all plates passed this quality check. A siRNA was a hit when it resulted in a NPI of > 25% in at least 3 out of the 4 measurements. This analysis resulted in 51 siRNAs covering 47 genes (for four genes two siRNA constructs targeting the same gene were identified).
  • SSMD Strictly Standardized Mean Difference
  • ATPLite viability data were normalized to the average of the plate and 3 siRNA hits that caused > 30%> drop in viability in one out of the 2 independent runs were discarded, leading to a drop out of 2 genes.
  • a hit was considered to be on-target if at least one additional siRNA sequence different from the original adenoviral shRNA hit sequence is identified as a hit in the TSLP secretion assay. For that reason, sequences of siRNAs and shRNAs targeting the identified on-target genes were compared (data not shown). Except for one, none of the siRNA hit and shRNA hit sequences showed an overlap in sequence. The sequences were considered overlapping if they had overlapping target regions of at least 1 nucleotide in the TARGET mRNA sequence. Only one construct showed an overlap of 11 nucleotides.. Hence, the identified target genes are considered on-target. As this overlap is very minimal, this target is still considered to be on-target. The results for on target analysis are summarized in Table 5. Table 5. Results for on target analysis using siRNA
  • the different RV strains were tested at 3 dilutions ranging from 1/10 (highest concentration) up to the dilution that was needed for the RV serotype to induce full cytopathogenic effect in a HelaRh cell line.
  • the outcome of the profiling exercise on TSLP readout was the following: out of the 16 serotypes, only infection with RV59 and to a lesser extent RV2 induced TSLP secretion in donor 7 cells. Addition of IL-4 (1 ng/mL IL-4) did not further enhance the RV mediated TSLP release.
  • RV59 and RV2 have been confirmed in follow up experiments.
  • triggering of NHBE cells with respiratory syncitial virus (RSV) was also explored ( Figure 7), but RSV did not induce TSLP secretion.
  • the outcome of the profiling exercise on the cytokine mRNA expression was the following: out of the 16 different cytokines for which expression was analysed by qPCR, the expression of 10 of these (CXCL10, CXCL8, CCL5, CXCL5, CXCL2, CXCL3, CCL20, CXCL1, CSF2 and ⁇ ) was found to be up-regulated by rhinovirus infection, although not to the same extent by all rhinoviruses. The expression of these cytokines was also induced by polyLC trigger. The effect on the expression of those cytokines was not further measured and GM-CSF was chosen as an alternative read-out instead.
  • the secondary assay was performed using the same siRNA knock-down strategy as used in the on-target screen. All 3 SilencerSelect siRNA versions (Ambion) that were found on-target were tested. In addition, we also tested the 3 siRNA versions against 8 genes for which no positive siRNA was found in the siRNA on-target screen, but that were on-target in the rescreen, i.e. confirmed with two independent shRNA constructs.
  • NHBE donor 7 cells were transfected with siRNAs as described in Example 4 and subsequently triggered with 100 ⁇ /well undiluted RV59 for 1.5 h at 35°C. After removing the RV59 inoculum, cells were washed once with culture medium and cultured for another 48 h in this medium at 35°C. Supernatants of cells transfected with negative and positive siRNA controls were then subjected to GM-CSF/IP 10/CCL5 multiplex and TSLP MSD assays to decide on the final read out. Data of GM-CSF and TSLP are shown in Figure 8.
  • GM-CSF was used as read out for the secondary assay and GM-CSF levels in the supernatants of cells infected with RV59 after transfection with siRNAs targeting the on-target genes, was quantified.
  • Z-scoring method was applied which is based on all negative controls on each plate ( Figure 9). Genes were designated hits if they scored as biological duplicate below a threshold value (Av - 2*SD of all negative control siRNAs on the plate).
  • the results for the TARGETS are presented in Table 6 below. Table 6.
  • Table 6 The results of the secondary validation assay using siRNA
  • siRNA hits that were toxic were already excluded. In this section, we further wanted to eliminate that the inhibition of TSLP secretion by confirmed shRNAs were due to toxicity.
  • the hit shRNAs for all on-target confirmed genes were also tested in an ATPLite viability assay using NHBE cells from donor 4. In brief, cells were seeded and subsequently transduced with adenoviral shRNA for all on-target confirmed genes (see example 4) and viability was assessed 4 days after transduction using the ATPLite assay kit (Perkin Elmer) according to manufacturer's instructions. Data were subsequently expressed as % change in cell numbers relative to negative controls. shRNAs that induced a drop of more than 30% in cell numbers were considered toxic. All TARGETS described in Table 8 showed less than 30% drop in cell number, indicating that their inhibitory effect on TSLP and GM-CSF is not due to cytotoxic effects in NHBE cells.
  • Example 7 Profiling the effect of the TARGETS knock-down by synthetic siRNA approach on innate
  • IFNB1 mRNA expression levels were comparable between non- transfected and negative control transfected conditions for the 6 hour time point.
  • IFNB1 siRNAs partially blocked the IFN response. Inhibition of IFNB1 mRNA in these conditions was also observed with siRNA that targets RELA. Optimal conditions were selected and siRNAs (up to 3 single siRNAs per gene) targeting the identified gene targets were tested for their effect on pIC mediated IFNB1 mRNA expression. The test was performed in triplicate.
  • siRNA/INTERFERin complexation mix was prepared first prepared as follows: 10.5 ⁇ siRNA (from 2 ⁇ stock solution) was transferred to V-bottom 96-well plate and 1 14.5 ⁇ BEGM basal medium (w/o supplements) was added ; to this, 125 ⁇ (containing 1.25 ⁇ INTERFERin) was added, and after mixing, siRNA/INTERFERin complexation was allowed to take on for 15-30 minutes at room temperature. Next, 50 ⁇ of the siRNA/INTERFERin complexation mix was added to the cells.
  • Group A demonstrated 40%> or more up-regulation of ⁇ ; only inhibition of AHSG caused 50%> or more inhibition of ⁇ . Inhibition of expression of many other TARGETS did not significantly inhibit IFN production release by the cell (TARGETS in the group B).
  • Group C represents the group of TARGETS for which the measurements were not either performed or were not reliable to give an indication of the effect.
  • Table 7. Summary of the effect of siRNA KD of different targets and the effect of the KD on IFNp
  • TSLP an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation.
  • Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer The Journal of Experimental Medicine v. 208 no. 3 469-478

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Abstract

La présente invention concerne des procédés et des essais pour l'identification d'agents capables de réguler à la baisse des réponses Th2 dans des cellules, en particulier des agents inhibant des réponses Th2. Une telle inhibition est utile dans la prévention, l'amélioration et/ou le traitement de nombreuses maladies associées au déséquilibre des réponses Th1/Th2. En particulier, la présente invention concerne des procédés et des essais pour identifier des agents pour l'utilisation dans la prévention et/ou le traitement de l'asthme, de la rhinite allergique, de la dermatite atopique, de la fibrose, de l'athérosclérose, de la colite ulcéreuse, d'une maladie inflammatoire chronique de l'intestin, de la glomérulonéphrite, de la cryoglobulinémie, d'un déficit immunitaire, de la rhinoconjonctivite, de l'anaphylaxie et de cancers. L'invention concerne un polypeptide et des acides nucléiques cibles et des séquences ARNsi à base de ces cibles.
PCT/EP2013/074723 2012-11-30 2013-11-26 Cibles moléculaires et composés, et procédés d'identification de ceux-ci, utiles dans la régulation à la baisse de la réponse th2 WO2014082993A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017178869A1 (fr) * 2016-04-15 2017-10-19 Pontificia Universidad Católica De Chile Composition pharmaceutique pour diminuer les symptômes et la maladie de l'infection respiratoire causée par le métapneumovirus humain (hmpv) qui comprend au moins un agent neutralisant la fonction des molécules tspl et/ou tslpr, et/ou ox40l et/ou cd177, et un excipient pharmaceutiquement acceptable, et leur utilisation
WO2019145386A1 (fr) * 2018-01-26 2019-08-01 Roche Innovation Center Copenhagen A/S Oligonucléotides pour la modulation de l'expression de csnk1d
WO2024059336A1 (fr) * 2022-09-18 2024-03-21 Memorial Sloan Kettering Cancer Center Inhibition de la voie du récepteur 2 activé par la protéase (par2) pour le traitement et la prévention du lymphoedème

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* Cited by examiner, † Cited by third party
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AU1373099A (en) * 1997-11-13 1999-06-07 Napolitano, Monica Th2 cell depletion; compositions; methods
US6762341B2 (en) * 2000-02-10 2004-07-13 Schering Corporation Uses of mammalian CCR8 receptors and related reagents

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017178869A1 (fr) * 2016-04-15 2017-10-19 Pontificia Universidad Católica De Chile Composition pharmaceutique pour diminuer les symptômes et la maladie de l'infection respiratoire causée par le métapneumovirus humain (hmpv) qui comprend au moins un agent neutralisant la fonction des molécules tspl et/ou tslpr, et/ou ox40l et/ou cd177, et un excipient pharmaceutiquement acceptable, et leur utilisation
US11065301B2 (en) 2016-04-15 2021-07-20 Pontificia Universidad Catolica De Chile Pharmaceutical composition for ameliorating the symptoms and disease of the respiratory infection caused by human metapneumovirus (HMPV), which comprises at least one agent that neutralizes the function of TSLP and/or TSLPR and/or OX40L and/or CD177 molecules, and a pharmaceutically acceptable excipient, and use thereof
WO2019145386A1 (fr) * 2018-01-26 2019-08-01 Roche Innovation Center Copenhagen A/S Oligonucléotides pour la modulation de l'expression de csnk1d
WO2024059336A1 (fr) * 2022-09-18 2024-03-21 Memorial Sloan Kettering Cancer Center Inhibition de la voie du récepteur 2 activé par la protéase (par2) pour le traitement et la prévention du lymphoedème

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