WO2002012887A2 - Methods and compositions for the diagnosis and treatment of brown adipose cell disorders - Google Patents

Methods and compositions for the diagnosis and treatment of brown adipose cell disorders Download PDF

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WO2002012887A2
WO2002012887A2 PCT/US2001/024901 US0124901W WO0212887A2 WO 2002012887 A2 WO2002012887 A2 WO 2002012887A2 US 0124901 W US0124901 W US 0124901W WO 0212887 A2 WO0212887 A2 WO 0212887A2
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cide
brown adipose
modulator
activity
nucleic acid
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PCT/US2001/024901
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French (fr)
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WO2002012887A3 (en
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Ruth Gimeno
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Millennium Pharmaceuticals, Inc.
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Publication of WO2002012887A3 publication Critical patent/WO2002012887A3/en

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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
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • Obesity represents the most prevalent of body weight disorders with estimates ranging from 30% to 50% within the middle-aged population in the western world.
  • Other body weight disorders such as anorexia nervosa and bulimia nervosa which together affect approximately 0.2% of the female population of the western world, also pose serious health threats.
  • disorders as anorexia and cachexia (wasting) are also prominent features of other diseases such as cancer, cystic fibrosis, and AIDS.
  • Obesity defined as a body mass index (BMI) of 30 kg/ 2 m or more, also contributes to other diseases.
  • BMI body mass index
  • this disorder is responsible for increased incidences of diseases such as coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia and some cancers.
  • Obesity is a complex multifactorial chronic disease that develops from an interaction of genotype and the environment. The development of obesity involves social, behavioral, cultural, physiological, metabolic and genetic factors.
  • thermogenesis adipose tissue that stores and releases fat according to the nutritional needs of the animal. BAT burns fat, releasing the energy as heat through thermogenesis. BAT thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy expenditure in response to increases in caloric intake (Sears, LB. et al. (1996) Mol. Cell. Biol.
  • BAT is also the major site of thermogenesis in rodents and plays an important role in thermogenesis in human infants.
  • brown fat diminishes with age, but can be re-activated under certain conditions, such as prolonged exposure to cold, maintenance on a high fat diet and in the presence of noradrenaline producing tumors.
  • Programmed cell death occurs in both vertebrate and invertebrate species and is characterized by unique morphological alterations, such as cytoplasmic contraction and chromatin condensation, as well as by specific DNA cleavage into oligonucleosomal fragments.
  • Brown adipose tissue mass is thought to be determined by the balance between presursor differentiation and mature adipocyte cell death.
  • Recent studies have demonstrated the presence of apoptotic events in brown adipose tissue. It has been found that the rate of apoptosis in brown adipose mass dramatically decreased under conditions requiring expansion of brown adipose tissue mass ⁇ e.g.
  • CIDEs A novel class of apoptotic molecules called CIDEs (CIDE-A, CIDE-B and FSP-
  • the present invention provides methods and compositions for the diagnosis and treatment of brown adipose cell disorders.
  • the present invention is based, at least in part, on the discovery that the apoptotic CIDE-A gene. (for cell death-inducing DFF45- like effector A), is expressed at high levels in brown adipose tissue (BAT) (see Figure 1).
  • BAT brown adipose tissue
  • the CIDE-A molecules by participating in apoptosis, modulate brown adipose cell behavior and are useful as targets and therapeutic agents for the modulation of brown adipose cell activity, e.g., proliferation, and the treatment of brown adipose cell disorders.
  • the present invention provides methods for the diagnosis and treatment of diseases including but not limited to obesity, anorexia, cachexia, and diabetes.
  • the invention provides methods for identifying a compound capable of treating a brown adipose cell disorder, e.g. , obesity, anorexia, or cachexia.
  • the method includes assaying the ability of the compound to modulate CIDE-A nucleic acid expression or CIDE-A polypeptide activity.
  • the ability of the compound to modulate nucleic acid expression or CIDE-A polypeptide activity is determined by detecting apoptosis of a brown adipose cell.
  • the ability of the compound to nucleic acid expression or CIDE-A polypeptide activity is determined by detecting modulation of thermogenesis.
  • the invention provides methods for identifying a compound capable of modulating a brown adipose cell activity, e.g., cell proliferation, differentiation, or cell death.
  • the method includes contacting a cell expressing a CIDE- A nucleic acid or polypeptide ⁇ e.g. , a brown adipose cell) with a test compound and assaying the ability of the test compound to modulate the expression of a CIDE-A nucleic acid or the activity of a CIDE-A polypeptide.
  • Another aspect of the invention provides a method for modulating a brown adipose cell activity, e.g., cell proliferation, cell differentiation, or cell death.
  • the method includes contacting a brown adipose cell with a CIDE-A modulator, for example, an anti-CIDE-A antibody, a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof, a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, a small molecule, an antisense CIDE-A nucleic acid molecule, a nucleic acid molecule of SEQ ID NO:l or a fragment thereof, or a ribozyme.
  • a CIDE-A modulator for example, an anti-CIDE-A antibody, a CIDE-A polypeptide comprising the amino
  • the invention features a method for treating a subject having a brown adipose cell disorder characterized by aberrant CIDE-A polypeptide activity or aberrant CIDE-A nucleic acid expression, e.g., obesity, anorexia, or cachexia.
  • the method includes administering to the subject a CIDE-A modulator, e.g., in a pharmaceutically acceptable formulation or by using a gene therapy vector.
  • Embodiments of this aspect of the invention include the CIDE-A modulator being a small molecule, an anti-CIDE-A antibody, a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof, a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, an antisense CIDE- A nucleic acid molecule, a nucleic acid molecule of SEQ ID NO:l or a fragment thereof, or a ribozyme.
  • the invention provides a method for modulating, e.g. , increasing or decreasing, thermogenesis in a subject by administering to the subject a CIDE-A modulator.
  • the invention also provides a method for modulating brown adipose cell apoptosis in a subject by administering to the subject a CIDE-A modulator.
  • Figure 1A is a graph depicting the results of a TaqManTM analysis of CIDE-A cDNA expression in normal mouse tissues, including brown adipose tissue (BAT) and white adipose tissue (WAT).
  • BAT brown adipose tissue
  • WAT white adipose tissue
  • Figure IB is a graph depicting the results of a TaqManTM analysis of FSP-27 cDNA expression in normal mouse tissues, including brown adipose tissue (BAT) and white adipose tissue (WAT).
  • BAT brown adipose tissue
  • WAT white adipose tissue
  • the present invention provides methods and compositions for the diagnosis and treatment of brown adipose cell disorders.
  • the CIDE-A modulators identified according to the methods of the invention can be used to modulate apoptosis of brown adipose tissue (BAT) and are, therefore, useful in treating or diagnosing brown adipose cell disorders.
  • BAT brown adipose tissue
  • inhibition of the activity of a CIDE-A molecule can cause increased BAT mass and, therefore, increased thermogenesis in a subject, thereby promoting weight loss in the subject.
  • the CIDE-A modulators used in the methods of the of the invention can be used to treat obesity.
  • CIDE-A modulators can decrease BAT by increasing apoptosis of brown adipocytes, thus decreasing thermogenesis in a subject, thereby inhibiting weight loss in the subject.
  • CIDE-A modulators are also useful in the treatment of undesirable weight loss, e.g., cachexia or anorexia. Modulators of CIDE-A can also be effective in the treatment of diabetes caused by insulin resistance.
  • a "brown adipose cell disorder” includes a disease, disorder, or condition which affects a brown adipose cell or tissue.
  • Brown adipose cell disorders include diseases, disorders, or conditions associated with aberrant thermogenesis or aberrant brown adipose cell content or function.
  • Brown adipose cell disorders can be characterized by a misregulation ⁇ e.g., downregulation or upregulation) of CIDE-A activity.
  • brown adipose cell disorders include disorders such as obesity, overweight, anorexia, cachexia, and diabetes.
  • Obesity is defined as a body mass index (BMI) of 30 kg/ 2 m or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)).
  • the present invention is also intented to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg/ 2 m or more, 26 kg/ 2 m or more, 27 kg/ 2 m or more, 28 kg/ 2 m or more, 29 kg/ 2 m or more, 29.5 kg/ 2 m or more, or 29.9 kg/ 2 m or more, all of which are typically refered to as overweight
  • BMI body mass index
  • CIDE-A activity includes an activity exerted by a CIDE-A protein, polypeptide or nucleic acid molecule on a CIDE-A responsive cell or tissue, e.g., BAT, or on a CIDE-A protein substrate, as determined in vivo, or in vitro, according to standard techniques.
  • CIDE-A activity can be a direct activity, such as an association with a CIDE-A-target molecule.
  • a “substrate” or “target molecule” or “binding partner” is a molecule with which a CIDE-A protein binds or interacts in nature, such that CIDE-A-mediated function, e.g. , modulation of apoptosis, is achieved.
  • a CIDE-A target molecule can be a non-CIDE-A molecule or a CIDE-A protein or polypeptide. Examples of such target molecules include proteins in the same signaling path as the CIDE-A protein, e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) or downstream of the CIDE-A protein in a pathway involving regulation of brown adipose cell apoptosis.
  • a CIDE-A activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the CIDE-A protein with a CIDE-A target molecule.
  • the biological activities of CIDE-A are described herein.
  • the CIDE-A proteins can have one or more of the following activities: 1) they modulate apoptosis in brown adipose cells; 2) they modulate thermogenesis; and 3) they modulate insulin sensitivity.
  • brown adipose cell activity includes an activity exerted by a brown adipose cell, or an activity that takes place in a brown adipose cell.
  • acitivities include cellular processes that contribute to the physiological role of brown adipose cells, such as lipogenesis and lipolysis and include, but are not limited to, cell proliferation, differentiation, growth, migration, programmed cell death, uncoupled mitochondrial respiration, and thermogenesis.
  • the invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents ⁇ e.g., peptides, peptidomimetics, small molecules, ribozymes, or CIDE-A antisense molecules) which bind to CIDE-A proteins, have a stimulatory or inhibitory effect on CIDE-A expression or CIDE-A activity, or have a stimulatory or inhibitory effect on the expression or activity of a CIDE-A target molecule.
  • modulators i.e., candidate or test compounds or agents ⁇ e.g., peptides, peptidomimetics, small molecules, ribozymes, or CIDE-A antisense molecules
  • Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al.
  • antibodies e.g., polyclonal, monoclonal, humanized, anti- idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') 2 , Fab expression library fragments, and epitope-binding fragments of antibodies
  • small organic and inorganic molecules e.g., molecules obtained from combinatorial and natural product libraries.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • Assays that may be used to identify compounds that modulate CIDE-A activity include assays for cytochrome C release from mitochondria during cell apoptosis, e.g., brown fat cell apoptosis (as described in, for example, Bossy- Wetzel E. et al. (2000) Methods in Enzymol. 322:235-42); cytofluorometric quantitation of nuclear apoptosis induced in a cell-free system (as described in, for example, Lorenzo H.K. et al. (2000) Methods in Enzymol. 322:198-201); apoptotic nuclease assays (as described in, for example, Hughes F.M.
  • cytochrome C release from mitochondria during cell apoptosis e.g., brown fat cell apoptosis (as described in, for example, Bossy- Wetzel E. et al. (2000) Methods in Enzymol. 322:
  • apoptotic cells e.g., apoptotic brown fat cells
  • flow and laser scanning cytometry as described in, for example, Darzynkiewicz Z. et al. (2000) Methods in Enzymol. 322:18-39
  • detection of apoptosis by annexin V labeling as described in, for example, Bossy- Wetzel E. et al. (2000) Methods in Enzymol. 322:15-18
  • transient transfection assays for cell death genes as described in, for example, Miura M. et al. (2000) Methods in Enzymol.
  • apoptotic cells e.g., apoptotic brown fat cells (as described in, for example, Kauffman S.H. et al. (2000) Methods in Enzymol. 322:3-15).
  • an assay is a cell-based assay in which a cell which expresses a CIDE-A protein or biologically active portion thereof ⁇ e.g. , the N-terminal region (amino acid residues 1-107) of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity or the the C-terminal region (amino acid residues 108- 200) of the CIDE-A protein that is necessary for induction of apoptosis in cells) is contacted with a test compound and the ability of the test compound to modulate CIDE- A activity is determined.
  • the biologically active portion of the CIDE-A protein includes a domain or motif that can modulate apoptosis of brown adipose cells (adipocytes) and/or which can modulate thermogenesis. Determining the ability of the test compound to modulate CIDE-A activity can be accomplished by monitoring, for example, the production of one or more specific metabolites ⁇ e.g., ⁇ C glucose, see below) in a cell which expresses CIDE-A (see, e.g., Saada et al. (2000) Biochem Biophys. Res. Commun. 269: 382-386) or by monitoring cell death, cell proliferation, or cell differentiation in the cell.
  • adipocytes brown adipose cells
  • the cell for example, can be of mammalian origin, e.g., an adipose cell such as a brown adipose cell, an HIB-1B brown adipose tumor cell line, a 3T3-L1 adipogenic cell line, or a TA1 adipogenic cell line.
  • an adipose cell such as a brown adipose cell, an HIB-1B brown adipose tumor cell line, a 3T3-L1 adipogenic cell line, or a TA1 adipogenic cell line.
  • the ability of the test compound to modulate CIDE-A binding to a substrate or to bind to CIDE-A can also be determined. Determining the ability of the test compound to modulate CIDE-A binding to a substrate can be accomplished, for example, by coupling the CIDE-A substrate with a radioisotope or enzymatic label such that binding of the CIDE-A substrate to CIDE-A can be determined by detecting the labeled CIDE-A substrate in a complex. Alternatively, CIDE-A could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate CIDE-A binding to a CIDE-A substrate in a complex.
  • Determining the ability of the test compound to bind CIDE-A can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to CIDE-A can be determined by detecting the labeled CIDE-A compound in a complex.
  • CIDE-A substrates can be labeled with ⁇ 5 ⁇ 35s ; H or 3j ⁇ either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a compound to interact with CIDE-A without the labeling of any of the interactants.
  • a microphysiometer can be used to detect the interaction of a compound with CIDE-A without the labeling of either the compound or the CIDE-A (McConnell, H. M. et al. (1992) Science 257:1906-1912).
  • a "microphysiometer” ⁇ e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • the ability of a CIDE-A modulator to modulate, e.g., inhibit or increase, CIDE- A activity can also be determined through screening assays which identify modulators which either increase or decrease apoptosis and DNA fragmentation.
  • the invention provides for a screening assay involving contacting cells which express a CIDE-A protein or polypeptide with a test compound, and examining the cells for the morphological features of apoptosis. For example, cells expressing a CIDE-A protein or polypeptide can be contacted with a test compound and nuclearly stained with acridine orange. Subsequently, nuclear DNA can be extracted and analyzed for DNA fragmentation as described in Inohora et al, (1997) EMBO J.
  • CAT chloramphenicol acetyltransferase
  • CIDE-A expression can also be used to test the ability of the CIDE-A molecule to modulate adipogenesis, e.g., differentiation of white adipose tissue to brown adipose tissue, as CIDE-A expression is specific to brown adipose tissue. If a test compound can modulate CIDE-A expression is can most likely modulate the differentiation of white adipose tissue to brown adipose tissue.
  • the ability of a test compound to modulate the differentiation of white adipose tissue to brown adipose tissue can be measured by introducing a test compound into a cell, e.g., a white adipose cell, and measuring the number of mitochondria in the cell as compared to the number of mitochondria in a control cell which does not contain the test compound.
  • brown adipose cells are known to contain substantially greater numbers of mitochondria than white adipocytes
  • an increase or decrease in the number of mitochondria (or in a mitochondrial marker such as cytochrome c oxidase) in the test cell as compared to the control cell indicates that the test compound can modulate differentiation of white adipose tissue to brown adipose tissue or vice versa (as described in, for example, PCT publication No.WO 00/32215).
  • the ability of a test compound to modulate insulin sensitivity of a cell can be determined by performing an assay in which cells, e.g., brown adipose cells, are contacted with the test compound, e.g., transformed to express the test compound; incubated with radioactively labeled glucose ( ⁇ C glucose); and treated with insulin.
  • ⁇ C glucose radioactively labeled glucose
  • An increase or decrease in glucose in the cells containing the test compound as compared to the control cells indicates that the test compound can modulate insulin sensitivity of the cells.
  • the cells containing the test compound can be incubated with a radioactively labeled phosphate source ⁇ e.g., [32p]ATP) and treated with insulin.
  • Phosphorylation of proteins in the insulin pathway e.g., the insulin receptor
  • An increase or decrease in phosphorylation of a protein in the insulin pathway in cells containing the test compound as compared to the control cells indicates that the test compound can modulate insulin sensitivity of the cells.
  • an assay of the present invention is a cell-free assay in which a CIDE-A protein or biologically active portion thereof (e.g., the N-terminal region of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity) is contacted with a test compound and the ability of the test compound to bind to or to modulate ⁇ e.g., stimulate or inhibit) the activity of the CIDE- A protein or biologically active portion thereof is determined.
  • Preferred biologically active portions of the CIDE-A proteins to be used in assays of the present invention include fragments which participate in interactions with non-CIDE-A molecules, e.g., fragments with high surface probability scores.
  • Binding of the test compound to the CIDE-A protein can be determined either directly or indirectly as described above. Determining the ability of the CIDE-A protein to bind to a test compound can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
  • BIOA Biomolecular Interaction Analysis
  • SPR surface plasmon resonance
  • binding of a test compound to a CIDE-A protein, or interaction of a CIDE-A protein with a CIDE-A target molecule in the presence and absence of a test compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/CIDE-A fusion proteins or glutathione-S-transferase/target 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 or the test compound and either the non-adsorbed target protein or CIDE-A protein, and the mixture incubated under conditions conducive to complex formation ⁇ e.g., at physiological conditions for salt and pH).
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtitre plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or CIDE-A protein, and the mixture incubated under conditions conducive to complex formation ⁇ e.g., at physiological conditions for salt and pH).
  • the matrix is immobilized in the case of beads, and complex formation is determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of CIDE- A binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CIDE-A protein or a CIDE-A target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated CIDE-A protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art ⁇ e.g. , biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotin-NHS N- hydroxy-succinimide
  • Pierce Chemicals Pierce Chemicals, Rockford, IL
  • IL streptavidin-coated 96 well plates
  • antibodies which are reactive with CIDE-A protein or target molecules but which do not interfere with binding of the CIDE-A protein to its target molecule can be derivatized to the wells of the plate, and unbound target or CIDE-A protein is trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the CIDE-A protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CIDE-A protein or target molecule.
  • the CIDE-A protein or fragments thereof can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et ⁇ /. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • CIDE-A-binding proteins proteins which bind to or interact with CIDE-A
  • CIDE-A-binding proteins proteins which bind to or interact with CIDE-A
  • CIDE-A-binding proteins are also likely to be involved in the propagation of signals by the CIDE-A proteins or CIDE-A targets as, for example, downstream elements of a CIDE-A-mediated signaling pathway.
  • CIDE-A-binding proteins are likely to be CIDE-A inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a CIDE-A protein is fused to a gene encoding the DNA binding domain of a known transcription factor ⁇ e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene ⁇ e.g, LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CIDE-A protein.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the activity of a CIDE-A protein can be confirmed in vivo, e.g., in an animal such as an animal model for obesity, anorexia, or cachexia.
  • animals that can be used include the transgenic mouse decribed in U.S. Patent No. 5,932,779 that contains a mutation in an endogenous melanocortin-4-receptor (MC4-R) gene; animals having mutations which lead to syndromes that include obesity symptoms (described in, for example, Friedman, J. M. et al. (1991) Mamm. Gen. 1:130-144; Friedman, J. M.
  • mice null for the adipocyte fatty acid binding protein or the animals described in Loskutoff D.J. etal. (2000) Ann. N. Y. Acad. Sci. 902:272-81 (the fat mouse).
  • animals that may be used include non-recombinant, non- genetic animal models of obesity such as, for example, rabbit, mouse, or rat models in which the animal has been exposed to either prolonged cold or long-term over-eating, thereby, inducing hypertrophy of BAT and increasing BAT thermogenesis (Himms- Hagen, J. (1990), supra). Additionally, animals created by ablation of BAT through use of targeted expression of a toxin gene (Lowell, B. et al. (1993) Nature 366:740-742) may be used.
  • a CIDE-A modulator identified as described herein e.g., an antisense CIDE-A nucleic acid molecule, a CIDE-A-specific antibody, or a small molecule
  • a CIDE-A modulator identified as described herein can be used in an animal model to determine the mechanism of action of such a modulator.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining CIDE-A protein and/or nucleic acid expression as well as CIDE-A activity in the context of a biological sample ⁇ e.g., blood, serum, cells, or tissue, e.g., brown adipose tissue) to thereby determine whether an individual is afflicted with a brown adipose cell disorder.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a brown adipose cell disorder. For example, mutations in a CIDE-A gene can be assayed for in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a brown adipose cell disorder.
  • Another aspect of the invention pertains to monitoring the influence of CIDE-A modulators ⁇ e.g., anti-CIDE-A antibodies or CIDE-A ribozymes) on the expression or activity of CIDE-A in clinical trials.
  • CIDE-A modulators e.g., anti-CIDE-A antibodies or CIDE-A ribozymes
  • a biological sample may be obtained from a subject and the biological sample may be contacted with a compound or an agent capable of detecting a CIDE-A protein or nucleic acid (e.g., mRNA or genomic DNA) that encodes a CIDE-A protein, in the biological sample.
  • a preferred agent for detecting CIDE-A mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to CIDE-A mRNA or genomic
  • the nucleic acid probe can be, for example, the CIDE-A nucleic acid set forth in SEQ ID NO:l, or a portion thereof, such as an oligonucleotide of at least 15, 20, 25, 30, 25, 40, 45, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to CIDE-A mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting CIDE-A protein in a sample is an antibody capable of binding to CIDE-A protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • an intact antibody, or a fragment thereof can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. That is, the detection method of the invention can be used to detect CIDE-A mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of CIDE-A mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of CIDE-A protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of CIDE-A genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of CIDE-A protein include introducing into a subject a labeled anti-CIDE-A antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting CIDE-A protein, mRNA, or genomic DNA, such that the presence of CIDE-A protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of CIDE-A protein, mRNA or genomic DNA in the control sample with the presence of CIDE-A protein, mRNA or genomic DNA in the test sample.
  • the present invention further pertains to methods for identifying subjects having or at risk of developing a brown adipose cell disorder associated with aberrant CIDE-A expression or activity.
  • aberrant includes a CIDE-A expression or activity which deviates from the wild type CIDE-A expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant CIDE-A expression or activity is intended to include the cases in which a mutation in the CIDE-A gene causes the CIDE-A gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional CIDE-A protein or a protein which does not function in a wild-type fashion, e.g. , a protein which does not interact with a CIDE-A substrate, or one which interacts with a non-CIDE-A substrate.
  • the assays described herein can be used to identify a subject having or at risk of developing a brown adipose cell disorder, e.g. , obesity, anorexia, cachexia, or diabetes.
  • a biological sample may be obtained from a subject and tested for the presence or absence of a genetic alteration.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a CIDE-A gene, 2) an addition of one or more nucleotides to a CIDE-A gene, 3) a substitution of one or more nucleotides of a CIDE-A gene, 4) a chromosomal rearrangement of a
  • CIDE-A gene 5) an alteration in the level of a messenger RNA transcript of a CIDE-A gene, 6) aberrant modification of a CIDE-A gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a CIDE-A gene, 8) a non-wild type level of a CIDE-A-protein, 9) allelic loss of a CIDE-A gene, and 10) inappropriate post-translational modification of a CIDE-A-protein.
  • a genetic alteration in a CIDE-A gene may be detected using a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran etal (1988) Science 241:1077-1080; andNakazawa et al. (1994) Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method includes collecting a biological sample from a subject, isolating nucleic acid (e.g., genomic DNA, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a CIDE-A gene under conditions such that hybridization and amplification of the CIDE-A gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic DNA, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. etal. ⁇ 1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a CIDE-A gene from a biological sample can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in CIDE-A can be identified by hybridizing biological sample derived and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M.T. et al. (1996) Human Mutation 7:244-255; Kozal, MJ. et al. (1996) Nature Medicine 2:753-759).
  • genetic mutations in CIDE-A can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. (1996) supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows for the identification of point mutations. This step is followed by a second hybridization array that allows for the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild- type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the CIDE-A gene in a biological sample and detect mutations by comparing the sequence of the CIDE-A in the biological sample with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W.
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the CIDE-A gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type CIDE-A sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397 and Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in CIDE-A cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes proteins that recognize mismatched base pairs in double-stranded DNA
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis
  • a probe based on a CIDE-A sequence e.g., a wild-type CIDE-A sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in CIDE-A genes.
  • single strand conformation polymorphism SSCP
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control CIDE-A nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the prognostic assays described herein can be used to determine whether a subject can be administered a CIDE-A modulator (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, or small molecule) to effectively treat a brown adipose cell disorder.
  • a CIDE-A modulator e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, or small molecule
  • the present invention further provides methods for determining the effectiveness of a CIDE-A modulator (e.g. , a CIDE-A modulator identified herein) in treating a brown adipose cell disorder in a subject.
  • a CIDE-A modulator e.g. , a CIDE-A modulator identified herein
  • the effectiveness of a CIDE-A modulator in increasing CIDE-A gene expression, protein levels, or in upregulating CIDE-A activity can be monitored in clinical trials of subjects exhibiting decreased CIDE-A gene expression, protein levels, or downregulated CIDE-A activity.
  • the effectiveness of a CIDE-A modulator in decreasing CIDE-A gene expression, protein levels, or in downregulating CIDE-A activity can be monitored in clinical trials of subjects exhibiting increased CIDE-A gene expression, protein levels, or CIDE-A activity.
  • a CIDE-A gene and preferably, other genes that have been implicated in, for example, a brown adipose cell disorder can be used as a "read out" or marker of the phenotype of a particular cell.
  • genes, including CIDE-A that are modulated in cells by treatment with an agent which modulates CIDE-A activity (e.g. , identified in a screening assay as described herein) can be identified.
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of CIDE-A and other genes implicated in the brown adipose cell disorder.
  • the levels of gene expression e.g., a gene expression pattern
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods described herein, or by measuring the levels of activity of CIDE-A or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent which modulates CIDE-A activity.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent which modulates CIDE-A activity (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, or small molecule identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a CIDE-A protein, mRNA, or genomic DNA in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the CIDE-A protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the CIDE-A protein, mRNA, or genomic DNA in the pre-administration sample with the CIDE
  • an agent which modulates CIDE-A activity e.g., an agonist, antagonist, peptidomimetic, protein,
  • increased administration of the agent may be desirable to increase the expression or activity of CIDE-A to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of CIDE-A to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • CIDE-A expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject, e.g., a human, at risk of (or susceptible to) a brown adipose cell disorder such as obesity, anorexia, cachexia, or diabetes.
  • a subject e.g., a human
  • a brown adipose cell disorder such as obesity, anorexia, cachexia, or diabetes.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • Another aspect of the invention provides methods for tailoring an subject's prophylactic or therapeutic treatment with either the CIDE-A molecules of the present invention or CIDE-A modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a brown adipose cell disorder by administering to the subject an agent which modulates CIDE-A expression or CIDE-A activity, e.g., modulation of adipose cell proliferation or modulation of apoptosis in adipose cells, e.g., brown adipose cells.
  • an agent which modulates CIDE-A expression or CIDE-A activity e.g., modulation of adipose cell proliferation or modulation of apoptosis in adipose cells, e.g., brown adipose cells.
  • Subjects at risk for a brown adipose cell disorder can be identified by, for example, any or a combination of the diagnostic or prognostic assays described herein.
  • a prophylactic agent can occur prior to the manifestation of symptoms characteristic of aberrant CIDE- A expression or activity, such that a brown adipose cell disorder is prevented or, alternatively, delayed in its progression.
  • a CIDE-A, CIDE-A agonist or CIDE-A antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • Another aspect of the invention pertains to methods for treating a subject suffering from a brown adipose cell disorder. These methods involve administering to a subject an agent which modulates CIDE-A expression or activity (e.g., an agent identified by a screening assay described herein), or a combination of such agents. In another embodiment, the method involves administering to a subject a CIDE-A protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted CIDE-A expression or activity.
  • an agent which modulates CIDE-A expression or activity e.g., an agent identified by a screening assay described herein
  • the method involves administering to a subject a CIDE-A protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted CIDE-A expression or activity.
  • Stimulation of CIDE-A activity is desirable in situations in which CIDE-A is abnormally downregulated and/or in which increased CIDE-A activity is likely to have a beneficial effect, i.e., an increase in induction of apoptosis in brown adipose cells, and a decrease in thermogenesis, thereby ameliorating brown adipose cell disorders such as anorexia or cachexia in a subject.
  • inhibition of CIDE-A activity is desirable in situations in which CIDE-A is abnormally upregulated and/or in which decreased CIDE-A activity is likely to have a beneficial effect, e.g., inhibition of apoptosis in brown adipose cells and an increase in thermogenesis, thereby ameliorating a brown adipose cell disorder such as obesity in a subject.
  • compositions suitable for such administration typically comprise the agent (e.g., nucleic acid molecule, protein, or antibody) and a pharmaceutically acceptable carrier.
  • agent e.g., nucleic acid molecule, protein, or antibody
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition used in the therapeutic methods of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the agent that modulates CIDE-A activity (e.g., a fragment of a CIDE-A protein or an anti-CIDE-A antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the agents that modulate CIDE-A activity can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the agents that modulate CIDE-A activity are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as efhylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the agent that modulates CIDE-A activity and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an agent for the treatment of subjects.
  • Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Agents which exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such CIDE-A modulating agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 ⁇ i. e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g. , about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • an animal e.g., a human
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 inter
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules used in the methods of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • pharmacogenomics i.e., the study of the relationship between a subject's genotype and that subject's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an agent which modulates CIDE-A activity, as well as tailoring the dosage and/or therapeutic regimen of treatment with an agent which modulates CIDE-A activity.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol. Physiol. 23(10-11): 983-985 andLinder, M.W. etal. (1997) Clin. Chem.43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate aminopeptidase deficiency
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants).
  • gene-related markers e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/TII drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNPs single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome.
  • treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach" can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug target is known (e.g., a CIDE-A protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and the cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6- formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., a CIDE-A molecule or CIDE-A modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of a subject. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and, thus, enhance therapeutic or prophylactic efficiency when treating a subject suffering from a brown adipose cell disorder with an agent which modulates CIDE-A activity.
  • the methods of the invention include the use of vectors, preferably expression vectors, containing a nucleic acid encoding a CIDE-A protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is a type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors to be used in the methods of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CIDE-A proteins, mutant forms of CIDE-A proteins, fusion proteins, and the like).
  • the recombinant expression vectors to be used in the methods of the invention can be designed for expression of CIDE-A proteins in prokaryotic or eukaryotic cells.
  • CIDE-A proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel (1990) supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a roteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • fusion proteins can be utilized in CIDE-A activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for CIDE-A proteins.
  • CIDE-a fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J. et al.,
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • the methods of the invention may further use a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) ofan RNA molecule which is antisense to CIDE-A mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to the use of host cells into which a CIDE-A nucleic acid molecule of the invention is introduced, e.g., a CIDE-A nucleic acid molecule within a recombinant expression vector or a CIDE-A nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a CIDE-a protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A
  • a host cell used in the methods of the invention can be used to produce ⁇ i.e., express) a CIDE-A protein.
  • the invention further provides methods for producing a CIDE-A protein using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a CIDE-A protein has been introduced) in a suitable medium such that a CIDE-A protein is produced.
  • the method further comprises isolating a CIDE-A protein from the medium or the host cell.
  • the coding sequence of the isolated human CIDE-A cDNA and the predicted amino acid sequence of the human CIDE-A polypeptide are shown in SEQ ID NOs:l and 2, respectively.
  • the CIDE-A sequence is also described in Inohara, et al. (1998), supra), the contents of which are incorporated herein by reference.
  • the methods of the invention include the use of isolated nucleic acid molecules that encode CIDE-A proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify CIDE-A-encoding nucleic acid molecules (e.g., CIDE-A mRNA) and fragments for use as PCR primers for the amplification or mutation of CIDE-A nucleic acid molecules.
  • CIDE-A-encoding nucleic acid molecules e.g., CIDE-A mRNA
  • fragments for use as PCR primers for the amplification or mutation of CIDE-A nucleic acid molecules e.g., CIDE-A mRNA
  • the term "nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-
  • a nucleic acid molecule used in the methods of the present invention e.g. , a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:l as a hybridization probe, CIDE-A nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1.
  • a nucleic acid used in the methods of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • oligonucleotides corresponding to CIDE-A nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
  • the isolated nucleic acid molecules used in the methods of the invention comprise the nucleotide sequence shown in SEQ ID NO:l, a complement of the nucleotide sequence shown in SEQ ID NO:l, or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO: 1, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l thereby forming a stable duplex.
  • an isolated nucleic acid molecule used in the methods of the present invention comprises a nucleotide sequence which is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l or a portion of any of this nucleotide sequence.
  • nucleic acid molecules used in the methods of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a CIDE-A protein, e.g., a biologically active portion of a CIDE-A protein.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO: 1 of an anti-sense sequence of SEQ ID NO : 1 or of a naturally occurring allelic variant or mutant of SEQ ID NO: 1.
  • a nucleic acid molecule used in the methods of the present invention comprises a nucleotide sequence which is greater than 100, 100-200, 200-300, 300-400, 400-500, 500-600, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
  • stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C.
  • SSC sodium chloride/sodium citrate
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45° C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present invention.
  • SSPE lxSSPE is 0.15M NaCl, lOmM NaH 2 P0 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g. , EDTA), Ficoll, PVP and the like.
  • blocking agents e.g., BSA or salmon or herring sperm carrier DNA
  • detergents e.g., SDS
  • chelating agents e.g. , EDTA
  • Ficoll e.g., Ficoll, PVP and the like.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CIDE-A protein, such as by measuring a level of a CIDE-A-encoding nucleic acid in a sample of cells from a subject e.g., detecting CIDE-A mRNA levels or determining whether a genomic CIDE-A gene has been mutated or deleted.
  • the methods of the invention further encompass the use of nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l due to degeneracy of the genetic code and thus encode the same CIDE-A proteins as those encoded by the nucleotide sequence shown in SEQ ID NO: 1.
  • an isolated nucleic acid molecule included in the methods of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
  • the methods of the invention further include the use of allelic variants of human CIDE-A, e.g. , fuctional and non-functional allelic variants.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human CIDE-A protein that maintain a CIDE-A activity. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the human CIDE-A protein that do not have a CIDE-A activity.
  • Non-functional allelic variants will typically contain a non-conservative substitution, deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion or deletion in critical residues or critical regions of the protein.
  • the methods of the present invention may further use non-human orthologues of the human CIDE-A protein.
  • Orthologues of the human CIDE-A protein are proteins that are isolated from non-human organisms and possess the same CIDE-A activity.
  • the methods of the present invention further include the use of nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l or a portion thereof, in which a mutation has been introduced.
  • the mutation may lead to amino acid substitutions at "non-essential” amino acid residues or at "essential” amino acid residues.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of CIDE-A (e.g., the sequence of SEQ ID NO: 2) without altering the biological activity, whereas an "essential” amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the CIDE-A proteins of the present invention and other members of the CIDE family e.g., CIDE-B, FSP-27, and DFF45 are not likely to be amenable to alteration.
  • Mutations can be introduced into SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains ⁇ e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains ⁇ e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a predicted nonessential amino acid residue in a CIDE-A protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a CIDE-A coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CIDE-A biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1 the encoded protein can be expressed recombinantly and the activity of the protein can be determined using the assay described herein.
  • Another aspect of the invention pertains to the use of isolated nucleic acid molecules which are antisense to the nucleotide sequence of SEQ ID NO: 1.
  • An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire CIDE-A coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a CIDE-A.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding CIDE-A.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of CIDE-A mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of CIDE-A mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CIDE-A mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules used in the methods of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CIDE-A protein to thereby inhibit expression of the protein, e.g. , by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g. , by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule used in the methods of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue etal.
  • an antisense nucleic acid used in the methods of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CIDE-A mRNA transcripts to thereby inhibit translation of CIDE-A mRNA.
  • a ribozyme having specificity for a CIDE-A-encoding nucleic acid can be designed based upon the nucleotide sequence of a CIDE-A cDNA disclosed herein (i.e., SEQ ID NO:l).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CIDE-A-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • CIDE-A mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • CIDE-A gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CIDE-A (e.g., the CIDE-A promoter and/or enhancers) to form triple helical structures that prevent transcription of the CIDE-A gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the CIDE-A e.g., the CIDE-A promoter and/or enhancers
  • the CIDE-A promoter and/or enhancers e.g., the CIDE-A promoter and/or enhancers
  • the CIDE-A nucleic acid molecules used in the methods of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93 : 14670-675.
  • PNAs of CIDE-A nucleic acid molecules can be used in the therapeutic and diagnostic applications described herein.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of CIDE-A nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) supra).
  • PNAs of CIDE-A can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of CIDE-A nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g. , RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. et al. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. et al. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • modified nucleoside analogs e.g., 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119- 11124).
  • the oligonucleotide used in the methods of the invention may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • another molecule e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent.
  • the methods of the invention include the use of isolated CIDE-A proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-CIDE-A antibodies.
  • native CIDE-A proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • CIDE-A proteins are produced by recombinant DNA techniques.
  • a CIDE-A protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • a "biologically active portion" of a CIDE-A protein includes a fragment of a CIDE-A protein having a CIDE-A activity.
  • Biologically active portions of a CIDE-A protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the CIDE-A protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include fewer amino acids than the full length CIDE-A proteins, and exhibit at least one activity of a CIDE-A protein.
  • biologically active portions comprise a domain or motif with at least one activity of the CIDE-A protein (e.g., the N-terminal region of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity).
  • a biologically active portion of a CIDE-A protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300 or more amino acids in length.
  • Biologically active portions of a CIDE-A protein can be used as targets for developing agents which modulate a CIDE-A activity.
  • the CIDE-A protein used in the methods of the invention has an amino acid sequence shown in SEQ ID NO:2.
  • the CIDE-A protein is substantially identical to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection V above.
  • the CIDE-A protein used in the methods of the invention is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the CIDE-A amino acid sequence of SEQ ID NO:2 having 500 amino acid residues, at least 75, preferably at least 150, more preferably at least 225, even more preferably at least 300, and even more preferably at least 400 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ⁇ J. Mol Biol 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a-NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller ⁇ Comput. Appl Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • CIDE-A chimeric or fusion proteins may also use CIDE-A chimeric or fusion proteins.
  • a CIDE-A "chimeric protein” or “fusion protein” comprises a CIDE-A polypeptide operatively linked to a non-CIDE-A polypeptide.
  • An "CIDE-A polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a CIDE-A molecule
  • a “non-CIDE-A polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the CIDE-A protein, e.g., a protein which is different from the CIDE-A protein and which is derived from the same or a different organism.
  • a CIDE-A fusion protein the CIDE-A polypeptide can correspond to all or a portion of a CIDE-A protein.
  • a CIDE-A fusion protein comprises at least one biologically active portion of a CIDE-A protein.
  • a CIDE-A fusion protein comprises at least two biologically active portions of a CIDE-A protein.
  • the term "operatively linked" is intended to indicate that the CIDE-A polypeptide and the non-CIDE-A polypeptide are fused in-frame to each other.
  • the non-CIDE-A polypeptide can be fused to the N-terminus or C-terminus of the CIDE-A polypeptide.
  • the fusion protein is a GST-CIDE-A fusion protein in which the CIDE-A sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant CIDE-A.
  • this fusion protein is a CIDE-A protein containing a heterologous signal sequence at its N-tenninus.
  • CIDE-A protein containing a heterologous signal sequence at its N-tenninus.
  • expression and/or secretion of CIDE-A can be increased through use of a heterologous signal sequence.
  • the CIDE-A fusion proteins used in the methods of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the CIDE-A fusion proteins can be used to affect the bioavailability of a CIDE-A substrate.
  • Use of CIDE-A fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a CIDE-A protein; (ii) mis-regulation of the CIDE-A gene; and (iii) aberrant post-translational modification of a CIDE-A protein.
  • the CIDE-A-fusion proteins used in the methods of the invention can be used as immunogens to produce anti-CIDE-A antibodies in a subject, to purify CIDE- A ligands and in screening assays to identify molecules which inhibit the interaction of CIDE-A with a CIDE-A substrate.
  • a CIDE-A chimeric or fusion protein used in the methods of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a CIDE-A-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CIDE-A protein.
  • the present invention also pertains to the use of variants of the CIDE-A proteins which function as either CIDE-A agonists (mimetics) or as CIDE-A antagonists.
  • Variants of the CIDE-A proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a CIDE-A protein.
  • An agonist of the CIDE-A proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a CIDE-A protein.
  • An antagonist of a CIDE-A protein can inhibit one or more of the activities of the naturally occurring form of the CIDE-A protein by, for example, competitively modulating a CIDE-A-mediated activity of a CIDE-A protein.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the CIDE-A protein.
  • variants of a CIDE-A protein which function as either
  • CIDE-A agonists or as CIDE-A antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of a CIDE-A protein for CIDE-A protein agonist or antagonist activity.
  • a variegated library of CIDE-A variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of CIDE-A variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CIDE-A sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. , for phage display) containing the set of CIDE-A sequences therein.
  • a set of larger fusion proteins e.g. , for phage display
  • CIDE-A variants from a degenerate oligonucleotide sequence Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CIDE-A sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et ⁇ /. (1984) Annu. Rev. Biochem. 53:323; Itakura et ⁇ . (1984) Scz ' ewce 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
  • libraries of fragments of a CIDE-A protein coding sequence can be used to generate a variegated population of CIDE-A fragments for screening and subsequent selection of variants of a CIDE-A protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CIDE-A coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the CIDE-A protein.
  • Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CIDE-A proteins.
  • the most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • Recursive ensemble mutagenesis (REM) a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CIDE-A variants (Arkin and Yourvan (1992) Proc. Natl Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • the methods of the present invention further include the use of anti-CIDE-A antibodies.
  • An isolated CIDE-A protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind CIDE-A using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length CIDE-A protein can be used or, alternatively, antigenic peptide fragments of CIDE-A can be used as immunogens.
  • the antigenic peptide of CIDE-A comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of CIDE-A such that an antibody raised against the peptide forms a specific immune complex with the CIDE-A protein.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of CIDE-A that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • a CIDE-A immunogen is typically used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed CIDE-A protein or a chemically synthesized CIDE-A polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CIDE-A preparation induces a polyclonal anti-CIDE-A antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as a CIDE-A.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind CIDE-A molecules.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CIDE-A.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular CIDE-A protein with which it immunoreacts.
  • Polyclonal anti-CIDE-A antibodies can be prepared as described above by immunizing a suitable subject with a CIDE-A immunogen.
  • the anti-CIDE-A antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CIDE-A.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against CIDE-A can be isolated from the mammal (e.g. , from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds CIDE-A.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind CIDE-A, e.g., using a standard ELISA assay.
  • a monoclonal anti-CIDE-A antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CIDE-A to thereby isolate immunoglobulin library members that bind CIDE-A.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No.
  • recombinant anti-CIDE-A antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the methods of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No.
  • An anti-CIDE-A antibody can be used to detect CIDE-A protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the CIDE-A protein.
  • Anti-CIDE-A antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling ⁇ i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • This example describes the tissue distribution of CIDE-A and FSP-27 cDNA in normal mouse tissues.
  • Tissues were collected from 7 week old female C57/B16 mice.
  • RNA was prepared using the trizol method and treated with DNAse to remove contaminating genomic DNA.
  • cDNA was synthesized using standard techniques. Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control GAPDH gene confirming efficient removal of genomic DNA contamination.
  • the following primers (based on the mouse CIDE-A sequence) were used for TaqmanTM (PE Applied Biosystems) analysis:
  • CIDE-A forward primer (SEQ ID NO:3): 5'CCAGCTCGCCCTTTTCG 3' CIDE-A reverse primer (SEQ ID NO:4): 5' TGCTGGCCATCACCCC 3' CIDE-1 probe (SEQ ID NO:5): 5' TCAAACCATGACCGAAGTAGCCGGC 3' FSP-27 forward primer (SEQ ID NO:6): 5' TCCTCCCAGCCTGTCTGAAG 3'
  • FSP-27 reverse primer SEQ ID NO:7): 5' CATCCATGGCCCAAGGAAG 3' FSP-27 probe (SEQ ID NO:8): 5' TGCTGCAATGAAGACCCGAGTCCTC 3'.
  • the Taqman TM system was used according to the manufacturer's directions. To allow standardization between different tissues, each sample contained two probes distinguished by different fluorescent labels, a probe for the gene of interest ⁇ e.g., CIDE- A), as well as a probe for GAPDH as an internal control.
  • the threshold values at which the PCR amplification started were determined using the manufacturer's software. PCR cycle number at threshold value was designated as CT. Relative expression was calculated as -((CTtest-CTGAPDH)tissue of interest - (CTtest-CTGAPDH)lowest expressing tissue in panel) c ,
  • tissue distribution For in situ analysis, various tissues, e.g. white and brown adipose tissues, were first frozen on dry ice. Ten-micrometer-thick sections of the tissues were postfixed with 4% formaldehyde in DEPC-treated IX phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0J M triethanolamine-HCl (pH 8.0).
  • various tissues e.g. white and brown adipose tissues, were first frozen on dry ice. Ten-micrometer-thick sections of the tissues were postfixed with 4% formaldehyde in DEPC-treated IX phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0J M triethanolamine-HCl (pH 8.0).
  • Hybridizations were performed with 35s-radiolabeled (5 X 10? cpm/ml) cRNA probes. Probes were incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01 % sheared salmon sperm DNA, 0.01 % yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55 °C.
  • SDS sodium dodecyl sulfate
  • slides were washed with 2X SSC. Sections were then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with 1 O ⁇ g of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides were then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA
  • Sections were then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4°C for 7 days before being developed and counter stained.
  • In situ hybridization results show that the mouse CIDE-A gene is expressed at high levels in E15.5 mouse embryo and postnatal day 1.5 mouse with strong specific signal localized to brown adipose tissue. Also, there is high expression of CIDE-A in murine adult brown adipose tissue. There are low levels of expression of CIDE-A in testis and pancreas. There is no detected expression of CIDE-A in murine adult white adipose tissue, nor is there detected expression in murine adult brain, spleen, lymph node, spinal cord, ovary kidney, adrenal gland, or heart.

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Abstract

The present invention relates to methods and compositions for the diagnosis and treatment of brown adipose cell disorders, includins, but not limited to, obesity, overweight, anorexia, or diabetes. The invention further provides methods for identifying a compound capable of treating a brown adipose cell disorder. The invention also provides methods for identifying a compound capable of modulating a brown adipose cell activity. Yet further, the invention provides a method for modulating a brown adipose cell activity. In addition, the invention provides a method for treating a subject having a brown adipose cell disorder characterized by aberrant CIDE-A polypeptide activity or aberrant CIDE-A nucleic acid expression. In another aspect, the invention provides methods for increasing thermogenesis in a subject and methods for modulating brown adipose cell apoptosis in a subject.

Description

METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF BROWN ADIPOSE CELL DISORDERS
Related Applications This application claims priority to U.S. Patent Application No. 09/634,956 filed on August 08, 2000, incorporated herein in its entirety by reference.
Background of the Invention
Obesity represents the most prevalent of body weight disorders with estimates ranging from 30% to 50% within the middle-aged population in the western world. Other body weight disorders, such as anorexia nervosa and bulimia nervosa which together affect approximately 0.2% of the female population of the western world, also pose serious health threats. Further, such disorders as anorexia and cachexia (wasting) are also prominent features of other diseases such as cancer, cystic fibrosis, and AIDS. Obesity, defined as a body mass index (BMI) of 30 kg/2m or more, also contributes to other diseases. For example, this disorder is responsible for increased incidences of diseases such as coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia and some cancers. (See, e.g., Nishina, P.M. et al. (1994), Metab. 3:554-558; Grundy, S.M. & Barnett, J.P. (1990), Dis. Mon. 36:641-731). Obesity is a complex multifactorial chronic disease that develops from an interaction of genotype and the environment. The development of obesity involves social, behavioral, cultural, physiological, metabolic and genetic factors.
Generally, obesity results when energy intake exceeds energy expenditure. Energy can be expended by thermogenesis. Vertebrates possess two distinct types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores and releases fat according to the nutritional needs of the animal. BAT burns fat, releasing the energy as heat through thermogenesis. BAT thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy expenditure in response to increases in caloric intake (Sears, LB. et al. (1996) Mol. Cell. Biol.
16(7):3410-3419). BAT is also the major site of thermogenesis in rodents and plays an important role in thermogenesis in human infants. In humans, and to a lesser extend rodents, brown fat diminishes with age, but can be re-activated under certain conditions, such as prolonged exposure to cold, maintenance on a high fat diet and in the presence of noradrenaline producing tumors.
Nearly all experimental rodent models of obesity are accompanied by diminished or defective BAT function, usually as the first symptom in the progression of obesity (Himms-Hagen, J. (1989) Prog. LipidRes. 28:67-115; Himms-Hagen, J. (1990) FASEB J. 4:2890-2898). In addition, ablation of BAT in transgenic mice by targeted expression of a toxin gene results in obesity (Lowell, B. et al. (1993) Nature 366:740-742). Also, transgenic mice with decreased brown fat have glucose intolerance and insulin resistance and have markedly enhanced susceptibility to diabetes (Lowell, et al. (1997) Annu. Rev. Med. 48:307-316). Thus, the growth and differentiation of brown adipocytes are key determinants in an animal's ability to maintain energy balance and prevent obesity (Sears, LB. et al. (1996) Mol. Cell. Biol. 16(7) :3410-3419).
Programmed cell death occurs in both vertebrate and invertebrate species and is characterized by unique morphological alterations, such as cytoplasmic contraction and chromatin condensation, as well as by specific DNA cleavage into oligonucleosomal fragments. Brown adipose tissue mass is thought to be determined by the balance between presursor differentiation and mature adipocyte cell death. Recent studies have demonstrated the presence of apoptotic events in brown adipose tissue. It has been found that the rate of apoptosis in brown adipose mass dramatically decreased under conditions requiring expansion of brown adipose tissue mass {e.g. exposure to the cold or treatment with noradrenaline), and increased under conditions where brown adipose tissue mass decreased, {e.g., readaption to higher temperatures after exposure to cold) (Lindquist and Rehnmark (1998) J. Biol. Chem 273:30147-30156). Therefore, apoptosis may play an important role in regulating brown adipose tissue mass in vivo. A novel class of apoptotic molecules called CIDEs (CIDE-A, CIDE-B and FSP-
27), which have homology to DFF45, has been described (Inohara et al. (1998) EMBO 17:2526-2533). As described in Inhohara et al. (1998), the C-terminal region of the CIDE-A molecule (amino acid residues 108-200) contains the effector domain, i.e., the domain necessary for induction of apoptosis in cells. The N-terminal region of the CIDE-A molecule (amino acid residues 1-107), which has homology to DFF45, contains a protein-protein interaction domain that is believed to be involved in the regulation of apoptotic acitivity.
Summary of the Invention The present invention provides methods and compositions for the diagnosis and treatment of brown adipose cell disorders. The present invention is based, at least in part, on the discovery that the apoptotic CIDE-A gene. (for cell death-inducing DFF45- like effector A), is expressed at high levels in brown adipose tissue (BAT) (see Figure 1). The CIDE-A molecules, by participating in apoptosis, modulate brown adipose cell behavior and are useful as targets and therapeutic agents for the modulation of brown adipose cell activity, e.g., proliferation, and the treatment of brown adipose cell disorders. Accordingly, the present invention provides methods for the diagnosis and treatment of diseases including but not limited to obesity, anorexia, cachexia, and diabetes.
In one aspect, the invention provides methods for identifying a compound capable of treating a brown adipose cell disorder, e.g. , obesity, anorexia, or cachexia. The method includes assaying the ability of the compound to modulate CIDE-A nucleic acid expression or CIDE-A polypeptide activity. In one embodiment, the ability of the compound to modulate nucleic acid expression or CIDE-A polypeptide activity is determined by detecting apoptosis of a brown adipose cell. In another embodiment, the ability of the compound to nucleic acid expression or CIDE-A polypeptide activity is determined by detecting modulation of thermogenesis.
In another aspect, the invention provides methods for identifying a compound capable of modulating a brown adipose cell activity, e.g., cell proliferation, differentiation, or cell death. The method includes contacting a cell expressing a CIDE- A nucleic acid or polypeptide {e.g. , a brown adipose cell) with a test compound and assaying the ability of the test compound to modulate the expression of a CIDE-A nucleic acid or the activity of a CIDE-A polypeptide.
Another aspect of the invention provides a method for modulating a brown adipose cell activity, e.g., cell proliferation, cell differentiation, or cell death. The method includes contacting a brown adipose cell with a CIDE-A modulator, for example, an anti-CIDE-A antibody, a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof, a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, a small molecule, an antisense CIDE-A nucleic acid molecule, a nucleic acid molecule of SEQ ID NO:l or a fragment thereof, or a ribozyme.
In yet another aspect, the invention features a method for treating a subject having a brown adipose cell disorder characterized by aberrant CIDE-A polypeptide activity or aberrant CIDE-A nucleic acid expression, e.g., obesity, anorexia, or cachexia. The method includes administering to the subject a CIDE-A modulator, e.g., in a pharmaceutically acceptable formulation or by using a gene therapy vector. Embodiments of this aspect of the invention include the CIDE-A modulator being a small molecule, an anti-CIDE-A antibody, a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof, a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, an antisense CIDE- A nucleic acid molecule, a nucleic acid molecule of SEQ ID NO:l or a fragment thereof, or a ribozyme.
In another aspect, the invention provides a method for modulating, e.g. , increasing or decreasing, thermogenesis in a subject by administering to the subject a CIDE-A modulator. The invention also provides a method for modulating brown adipose cell apoptosis in a subject by administering to the subject a CIDE-A modulator.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Brief Description of the Drawings
Figure 1A is a graph depicting the results of a TaqMan™ analysis of CIDE-A cDNA expression in normal mouse tissues, including brown adipose tissue (BAT) and white adipose tissue (WAT). The levels of CIDE-A in BAT are approximately 20-fold higher compared to the levels of CIDE-A in WAT.
Figure IB is a graph depicting the results of a TaqMan™ analysis of FSP-27 cDNA expression in normal mouse tissues, including brown adipose tissue (BAT) and white adipose tissue (WAT). The levels of FSP-27 in WAT are approximately 10-fold higher compared to the levels of FSP-27 in BAT.
Detailed Description of the Invention
The present invention provides methods and compositions for the diagnosis and treatment of brown adipose cell disorders. The CIDE-A modulators identified according to the methods of the invention can be used to modulate apoptosis of brown adipose tissue (BAT) and are, therefore, useful in treating or diagnosing brown adipose cell disorders. For example, inhibition of the activity of a CIDE-A molecule can cause increased BAT mass and, therefore, increased thermogenesis in a subject, thereby promoting weight loss in the subject. Thus, the CIDE-A modulators used in the methods of the of the invention can be used to treat obesity. Alternatively, CIDE-A modulators can decrease BAT by increasing apoptosis of brown adipocytes, thus decreasing thermogenesis in a subject, thereby inhibiting weight loss in the subject. Thus, CIDE-A modulators are also useful in the treatment of undesirable weight loss, e.g., cachexia or anorexia. Modulators of CIDE-A can also be effective in the treatment of diabetes caused by insulin resistance. As used herein, a "brown adipose cell disorder" includes a disease, disorder, or condition which affects a brown adipose cell or tissue. Brown adipose cell disorders include diseases, disorders, or conditions associated with aberrant thermogenesis or aberrant brown adipose cell content or function. Brown adipose cell disorders can be characterized by a misregulation {e.g., downregulation or upregulation) of CIDE-A activity. Examples of brown adipose cell disorders include disorders such as obesity, overweight, anorexia, cachexia, and diabetes. Obesity is defined as a body mass index (BMI) of 30 kg/2m or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)). However, the present invention is also intented to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg/2m or more, 26 kg/2m or more, 27 kg/2m or more, 28 kg/2m or more, 29 kg/2m or more, 29.5 kg/2m or more, or 29.9 kg/2m or more, all of which are typically refered to as overweight
(National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)).
As used interchangeably herein, "CIDE-A activity ' "biological activity of CIDE-A" or "functional activity of CIDE-A," includes an activity exerted by a CIDE-A protein, polypeptide or nucleic acid molecule on a CIDE-A responsive cell or tissue, e.g., BAT, or on a CIDE-A protein substrate, as determined in vivo, or in vitro, according to standard techniques. CIDE-A activity can be a direct activity, such as an association with a CIDE-A-target molecule. As used herein, a "substrate" or "target molecule" or "binding partner" is a molecule with which a CIDE-A protein binds or interacts in nature, such that CIDE-A-mediated function, e.g. , modulation of apoptosis, is achieved. A CIDE-A target molecule can be a non-CIDE-A molecule or a CIDE-A protein or polypeptide. Examples of such target molecules include proteins in the same signaling path as the CIDE-A protein, e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) or downstream of the CIDE-A protein in a pathway involving regulation of brown adipose cell apoptosis.
Alternatively, a CIDE-A activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the CIDE-A protein with a CIDE-A target molecule. The biological activities of CIDE-A are described herein. For example, the CIDE-A proteins can have one or more of the following activities: 1) they modulate apoptosis in brown adipose cells; 2) they modulate thermogenesis; and 3) they modulate insulin sensitivity.
As used herein, "brown adipose cell activity" includes an activity exerted by a brown adipose cell, or an activity that takes place in a brown adipose cell. For example, such acitivities include cellular processes that contribute to the physiological role of brown adipose cells, such as lipogenesis and lipolysis and include, but are not limited to, cell proliferation, differentiation, growth, migration, programmed cell death, uncoupled mitochondrial respiration, and thermogenesis. Various aspects of the invention are described in further detail in the following subsections:
I. Screening Assays:
The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents {e.g., peptides, peptidomimetics, small molecules, ribozymes, or CIDE-A antisense molecules) which bind to CIDE-A proteins, have a stimulatory or inhibitory effect on CIDE-A expression or CIDE-A activity, or have a stimulatory or inhibitory effect on the expression or activity of a CIDE-A target molecule. Compounds identified using the assays described herein may be useful for treating brown adipose cell disorders.
Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al.
(1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti- idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).
The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al.
(1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994) J Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int.
Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem.. 37:1233. Libraries of compounds may be presented in solution {e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et α/. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).
Assays that may be used to identify compounds that modulate CIDE-A activity include assays for cytochrome C release from mitochondria during cell apoptosis, e.g., brown fat cell apoptosis (as described in, for example, Bossy- Wetzel E. et al. (2000) Methods in Enzymol. 322:235-42); cytofluorometric quantitation of nuclear apoptosis induced in a cell-free system (as described in, for example, Lorenzo H.K. et al. (2000) Methods in Enzymol. 322:198-201); apoptotic nuclease assays (as described in, for example, Hughes F.M. (2000) Methods in Enzymol. 322:47-62); analysis of apoptotic cells, e.g., apoptotic brown fat cells, by flow and laser scanning cytometry (as described in, for example, Darzynkiewicz Z. et al. (2000) Methods in Enzymol. 322:18-39); detection of apoptosis by annexin V labeling (as described in, for example, Bossy- Wetzel E. et al. (2000) Methods in Enzymol. 322:15-18); transient transfection assays for cell death genes (as described in, for example, Miura M. et al. (2000) Methods in Enzymol. 322:480-92); and assays that detect DNA cleavage in apoptotic cells, e.g., apoptotic brown fat cells (as described in, for example, Kauffman S.H. et al. (2000) Methods in Enzymol. 322:3-15).
In one aspect, an assay is a cell-based assay in which a cell which expresses a CIDE-A protein or biologically active portion thereof {e.g. , the N-terminal region (amino acid residues 1-107) of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity or the the C-terminal region (amino acid residues 108- 200) of the CIDE-A protein that is necessary for induction of apoptosis in cells) is contacted with a test compound and the ability of the test compound to modulate CIDE- A activity is determined. In a preferred embodiment, the biologically active portion of the CIDE-A protein includes a domain or motif that can modulate apoptosis of brown adipose cells (adipocytes) and/or which can modulate thermogenesis. Determining the ability of the test compound to modulate CIDE-A activity can be accomplished by monitoring, for example, the production of one or more specific metabolites {e.g., ^ C glucose, see below) in a cell which expresses CIDE-A (see, e.g., Saada et al. (2000) Biochem Biophys. Res. Commun. 269: 382-386) or by monitoring cell death, cell proliferation, or cell differentiation in the cell. The cell, for example, can be of mammalian origin, e.g., an adipose cell such as a brown adipose cell, an HIB-1B brown adipose tumor cell line, a 3T3-L1 adipogenic cell line, or a TA1 adipogenic cell line.
The ability of the test compound to modulate CIDE-A binding to a substrate or to bind to CIDE-A can also be determined. Determining the ability of the test compound to modulate CIDE-A binding to a substrate can be accomplished, for example, by coupling the CIDE-A substrate with a radioisotope or enzymatic label such that binding of the CIDE-A substrate to CIDE-A can be determined by detecting the labeled CIDE-A substrate in a complex. Alternatively, CIDE-A could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate CIDE-A binding to a CIDE-A substrate in a complex. Determining the ability of the test compound to bind CIDE-A can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to CIDE-A can be determined by detecting the labeled CIDE-A compound in a complex. For example, CIDE-A substrates can be labeled with ^5^ 35s; H or 3j^ either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a compound to interact with CIDE-A without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with CIDE-A without the labeling of either the compound or the CIDE-A (McConnell, H. M. et al. (1992) Science 257:1906-1912). As used herein, a "microphysiometer" {e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and CIDE-A.
The ability of a CIDE-A modulator to modulate, e.g., inhibit or increase, CIDE- A activity can also be determined through screening assays which identify modulators which either increase or decrease apoptosis and DNA fragmentation. In one embodiment, the invention provides for a screening assay involving contacting cells which express a CIDE-A protein or polypeptide with a test compound, and examining the cells for the morphological features of apoptosis. For example, cells expressing a CIDE-A protein or polypeptide can be contacted with a test compound and nuclearly stained with acridine orange. Subsequently, nuclear DNA can be extracted and analyzed for DNA fragmentation as described in Inohora et al, (1997) EMBO J. 16:1686-1694. To determine whether a test compound modulates CIDE-A expression, in vitro transcriptional assays can be performed. To perform such an assay, the full length promoter and enhancer of CIDE-A can be linked to a reporter gene such as chloramphenicol acetyltransferase (CAT) and introduced into host cells. The same host cells can then be transfected with the test compound. The effect of the test compound can be measured by testing CAT activity and comparing it to CAT activity in cells which do not contain the test compound. An increase or decrease in CAT activity indicates a modulation of CIDE-A expression and is, therefore, an indicator of the ability of the test compound to modulate thermogenesis in adipose cells. The above described assay for testing the ability of a test compound to modulate
CIDE-A expression can also be used to test the ability of the CIDE-A molecule to modulate adipogenesis, e.g., differentiation of white adipose tissue to brown adipose tissue, as CIDE-A expression is specific to brown adipose tissue. If a test compound can modulate CIDE-A expression is can most likely modulate the differentiation of white adipose tissue to brown adipose tissue. Alternatively, the ability of a test compound to modulate the differentiation of white adipose tissue to brown adipose tissue can be measured by introducing a test compound into a cell, e.g., a white adipose cell, and measuring the number of mitochondria in the cell as compared to the number of mitochondria in a control cell which does not contain the test compound. As brown adipose cells are known to contain substantially greater numbers of mitochondria than white adipocytes, an increase or decrease in the number of mitochondria (or in a mitochondrial marker such as cytochrome c oxidase) in the test cell as compared to the control cell indicates that the test compound can modulate differentiation of white adipose tissue to brown adipose tissue or vice versa (as described in, for example, PCT publication No.WO 00/32215).
The ability of a test compound to modulate insulin sensitivity of a cell can be determined by performing an assay in which cells, e.g., brown adipose cells, are contacted with the test compound, e.g., transformed to express the test compound; incubated with radioactively labeled glucose ( ^C glucose); and treated with insulin. An increase or decrease in glucose in the cells containing the test compound as compared to the control cells indicates that the test compound can modulate insulin sensitivity of the cells. Alternatively, the cells containing the test compound can be incubated with a radioactively labeled phosphate source {e.g., [32p]ATP) and treated with insulin. Phosphorylation of proteins in the insulin pathway, e.g., the insulin receptor, can then be measured. An increase or decrease in phosphorylation of a protein in the insulin pathway in cells containing the test compound as compared to the control cells indicates that the test compound can modulate insulin sensitivity of the cells. In yet another embodiment, an assay of the present invention is a cell-free assay in which a CIDE-A protein or biologically active portion thereof (e.g., the N-terminal region of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity) is contacted with a test compound and the ability of the test compound to bind to or to modulate {e.g., stimulate or inhibit) the activity of the CIDE- A protein or biologically active portion thereof is determined. Preferred biologically active portions of the CIDE-A proteins to be used in assays of the present invention include fragments which participate in interactions with non-CIDE-A molecules, e.g., fragments with high surface probability scores. Binding of the test compound to the CIDE-A protein can be determined either directly or indirectly as described above. Determining the ability of the CIDE-A protein to bind to a test compound can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants {e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either CIDE-A or a CIDE-A target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a CIDE-A protein, or interaction of a CIDE-A protein with a CIDE-A target molecule in the presence and absence of a test compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/CIDE-A fusion proteins or glutathione-S-transferase/target 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 or the test compound and either the non-adsorbed target protein or CIDE-A protein, and the mixture incubated under conditions conducive to complex formation {e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix is immobilized in the case of beads, and complex formation is determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of CIDE- A binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CIDE-A protein or a CIDE-A target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated CIDE-A protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art {e.g. , biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which are reactive with CIDE-A protein or target molecules but which do not interfere with binding of the CIDE-A protein to its target molecule can be derivatized to the wells of the plate, and unbound target or CIDE-A protein is trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies reactive with the CIDE-A protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CIDE-A protein or target molecule.
In yet another aspect of the invention, the CIDE-A protein or fragments thereof {e.g. , the N-terminal region of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity) can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et α/. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with CIDE-A ("CIDE-A-binding proteins" or "CIDE-A-bp) and are involved in CIDE-A activity. Such CIDE-A-binding proteins are also likely to be involved in the propagation of signals by the CIDE-A proteins or CIDE-A targets as, for example, downstream elements of a CIDE-A-mediated signaling pathway. Alternatively, such CIDE-A- binding proteins are likely to be CIDE-A inhibitors.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a CIDE-A protein is fused to a gene encoding the DNA binding domain of a known transcription factor {e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a CIDE-A- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene {e.g, LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CIDE-A protein. In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the activity of a CIDE-A protein can be confirmed in vivo, e.g., in an animal such as an animal model for obesity, anorexia, or cachexia. Examples of animals that can be used include the transgenic mouse decribed in U.S. Patent No. 5,932,779 that contains a mutation in an endogenous melanocortin-4-receptor (MC4-R) gene; animals having mutations which lead to syndromes that include obesity symptoms (described in, for example, Friedman, J. M. et al. (1991) Mamm. Gen. 1:130-144; Friedman, J. M. and Liebel, R. L. (1992) Cell 69:217-220; Bray, G. A. (1992) Prog. Brain Res. 93:333-341; and Bray, G. A. (1989) Amer. J. Clin. Nutr. 5:891-902); the animals described in Stubdal H. et al. (2000) Mol. Cell Biol. 20(3):878-82 (the mouse tubby phenotype characterized by maturity- onset obesity); the animals described in Abadie J.M. et al. Lipids (2000) 35(6):613-20 (the obese Zucker rat (ZR), a genetic model of human youth-onset obesity and type 2 diabetes mellitus); the animals described in Shaughnessy S. et al. (2000) Diabetes 49(6):904-l 1 (mice null for the adipocyte fatty acid binding protein); or the animals described in Loskutoff D.J. etal. (2000) Ann. N. Y. Acad. Sci. 902:272-81 (the fat mouse). Other examples of animals that may be used include non-recombinant, non- genetic animal models of obesity such as, for example, rabbit, mouse, or rat models in which the animal has been exposed to either prolonged cold or long-term over-eating, thereby, inducing hypertrophy of BAT and increasing BAT thermogenesis (Himms- Hagen, J. (1990), supra). Additionally, animals created by ablation of BAT through use of targeted expression of a toxin gene (Lowell, B. et al. (1993) Nature 366:740-742) may be used.
Moreover, a CIDE-A modulator identified as described herein (e.g., an antisense CIDE-A nucleic acid molecule, a CIDE-A-specific antibody, or a small molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator. Alternatively, a CIDE-A modulator identified as described herein can be used in an animal model to determine the mechanism of action of such a modulator. II. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining CIDE-A protein and/or nucleic acid expression as well as CIDE-A activity, in the context of a biological sample {e.g., blood, serum, cells, or tissue, e.g., brown adipose tissue) to thereby determine whether an individual is afflicted with a brown adipose cell disorder. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a brown adipose cell disorder. For example, mutations in a CIDE-A gene can be assayed for in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a brown adipose cell disorder.
Another aspect of the invention pertains to monitoring the influence of CIDE-A modulators {e.g., anti-CIDE-A antibodies or CIDE-A ribozymes) on the expression or activity of CIDE-A in clinical trials.
These and other agents are described in further detail in the following sections.
A. Diagnostic Assays For Brown Adipose Cell Disorders To determine whether a subject is afflicted with a brown adipose cell disorder, a biological sample may be obtained from a subject and the biological sample may be contacted with a compound or an agent capable of detecting a CIDE-A protein or nucleic acid (e.g., mRNA or genomic DNA) that encodes a CIDE-A protein, in the biological sample. A preferred agent for detecting CIDE-A mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to CIDE-A mRNA or genomic
DNA. The nucleic acid probe can be, for example, the CIDE-A nucleic acid set forth in SEQ ID NO:l, or a portion thereof, such as an oligonucleotide of at least 15, 20, 25, 30, 25, 40, 45, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to CIDE-A mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein. A preferred agent for detecting CIDE-A protein in a sample is an antibody capable of binding to CIDE-A protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. That is, the detection method of the invention can be used to detect CIDE-A mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of CIDE-A mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of CIDE-A protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of CIDE-A genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of CIDE-A protein include introducing into a subject a labeled anti-CIDE-A antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting CIDE-A protein, mRNA, or genomic DNA, such that the presence of CIDE-A protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of CIDE-A protein, mRNA or genomic DNA in the control sample with the presence of CIDE-A protein, mRNA or genomic DNA in the test sample.
B. Prognostic Assays For Brown Adipose Cell Disorders
The present invention further pertains to methods for identifying subjects having or at risk of developing a brown adipose cell disorder associated with aberrant CIDE-A expression or activity.
As used herein, the term "aberrant" includes a CIDE-A expression or activity which deviates from the wild type CIDE-A expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant CIDE-A expression or activity is intended to include the cases in which a mutation in the CIDE-A gene causes the CIDE-A gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional CIDE-A protein or a protein which does not function in a wild-type fashion, e.g. , a protein which does not interact with a CIDE-A substrate, or one which interacts with a non-CIDE-A substrate.
The assays described herein, such as the preceding diagnostic assays or the following assays, can be used to identify a subject having or at risk of developing a brown adipose cell disorder, e.g. , obesity, anorexia, cachexia, or diabetes. A biological sample may be obtained from a subject and tested for the presence or absence of a genetic alteration. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a CIDE-A gene, 2) an addition of one or more nucleotides to a CIDE-A gene, 3) a substitution of one or more nucleotides of a CIDE-A gene, 4) a chromosomal rearrangement of a
CIDE-A gene, 5) an alteration in the level of a messenger RNA transcript of a CIDE-A gene, 6) aberrant modification of a CIDE-A gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a CIDE-A gene, 8) a non-wild type level of a CIDE-A-protein, 9) allelic loss of a CIDE-A gene, and 10) inappropriate post-translational modification of a CIDE-A-protein.
As described herein, there are a large number of assays known in the art which can be used for detecting genetic alterations in a CIDE-A gene. For example, a genetic alteration in a CIDE-A gene may be detected using a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran etal (1988) Science 241:1077-1080; andNakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in a CIDE-A gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method includes collecting a biological sample from a subject, isolating nucleic acid (e.g., genomic DNA, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a CIDE-A gene under conditions such that hybridization and amplification of the CIDE-A gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. etal. {1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In an alternative embodiment, mutations in a CIDE-A gene from a biological sample can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in CIDE-A can be identified by hybridizing biological sample derived and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M.T. et al. (1996) Human Mutation 7:244-255; Kozal, MJ. et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in CIDE-A can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. (1996) supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows for the identification of point mutations. This step is followed by a second hybridization array that allows for the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild- type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the CIDE-A gene in a biological sample and detect mutations by comparing the sequence of the CIDE-A in the biological sample with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W. (1995) Biotechniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in the CIDE-A gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type CIDE-A sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397 and Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in CIDE-A cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis
15:1657-1662). According to an exemplary embodiment, a probe based on a CIDE-A sequence, e.g., a wild-type CIDE-A sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in CIDE-A genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control CIDE-A nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). .It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered a CIDE-A modulator (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, or small molecule) to effectively treat a brown adipose cell disorder.
C. Monitoring of Effects During Clinical Trials
The present invention further provides methods for determining the effectiveness of a CIDE-A modulator (e.g. , a CIDE-A modulator identified herein) in treating a brown adipose cell disorder in a subject. For example, the effectiveness of a CIDE-A modulator in increasing CIDE-A gene expression, protein levels, or in upregulating CIDE-A activity, can be monitored in clinical trials of subjects exhibiting decreased CIDE-A gene expression, protein levels, or downregulated CIDE-A activity. Alternatively, the effectiveness of a CIDE-A modulator in decreasing CIDE-A gene expression, protein levels, or in downregulating CIDE-A activity, can be monitored in clinical trials of subjects exhibiting increased CIDE-A gene expression, protein levels, or CIDE-A activity. In such clinical trials, the expression or activity of a CIDE-A gene, and preferably, other genes that have been implicated in, for example, a brown adipose cell disorder can be used as a "read out" or marker of the phenotype of a particular cell. For example, and not by way of limitation, genes, including CIDE-A, that are modulated in cells by treatment with an agent which modulates CIDE-A activity (e.g. , identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents which modulate CIDE-A activity on subjects suffering from a brown adipose cell disorder in, for example, a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of CIDE-A and other genes implicated in the brown adipose cell disorder. The levels of gene expression (e.g., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods described herein, or by measuring the levels of activity of CIDE-A or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent which modulates CIDE-A activity. This response state may be determined before, and at various points during treatment of the individual with the agent which modulates CIDE-A activity. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent which modulates CIDE-A activity (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, or small molecule identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a CIDE-A protein, mRNA, or genomic DNA in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the CIDE-A protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the CIDE-A protein, mRNA, or genomic DNA in the pre-administration sample with the CIDE-A protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of CIDE-A to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of CIDE-A to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, CIDE-A expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
III. Methods of Treatment of Subjects Suffering From Brown Adipose Cell Disorders:
The present invention provides for both prophylactic and therapeutic methods of treating a subject, e.g., a human, at risk of (or susceptible to) a brown adipose cell disorder such as obesity, anorexia, cachexia, or diabetes. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics," as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype").
Thus, another aspect of the invention provides methods for tailoring an subject's prophylactic or therapeutic treatment with either the CIDE-A molecules of the present invention or CIDE-A modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects. A. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a brown adipose cell disorder by administering to the subject an agent which modulates CIDE-A expression or CIDE-A activity, e.g., modulation of adipose cell proliferation or modulation of apoptosis in adipose cells, e.g., brown adipose cells. Subjects at risk for a brown adipose cell disorder can be identified by, for example, any or a combination of the diagnostic or prognostic assays described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of aberrant CIDE- A expression or activity, such that a brown adipose cell disorder is prevented or, alternatively, delayed in its progression. Depending on the type of CIDE-A aberrancy, for example, a CIDE-A, CIDE-A agonist or CIDE-A antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
B. Therapeutic Methods
Another aspect of the invention pertains to methods for treating a subject suffering from a brown adipose cell disorder. These methods involve administering to a subject an agent which modulates CIDE-A expression or activity (e.g., an agent identified by a screening assay described herein), or a combination of such agents. In another embodiment, the method involves administering to a subject a CIDE-A protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted CIDE-A expression or activity.
Stimulation of CIDE-A activity is desirable in situations in which CIDE-A is abnormally downregulated and/or in which increased CIDE-A activity is likely to have a beneficial effect, i.e., an increase in induction of apoptosis in brown adipose cells, and a decrease in thermogenesis, thereby ameliorating brown adipose cell disorders such as anorexia or cachexia in a subject. Likewise, inhibition of CIDE-A activity is desirable in situations in which CIDE-A is abnormally upregulated and/or in which decreased CIDE-A activity is likely to have a beneficial effect, e.g., inhibition of apoptosis in brown adipose cells and an increase in thermogenesis, thereby ameliorating a brown adipose cell disorder such as obesity in a subject.
The agents which modulate CIDE-A activity can be administered to a subject using pharmaceutical compositions suitable for such administration. Such compositions typically comprise the agent (e.g., nucleic acid molecule, protein, or antibody) and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition used in the therapeutic methods of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the agent that modulates CIDE-A activity (e.g., a fragment of a CIDE-A protein or an anti-CIDE-A antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The agents that modulate CIDE-A activity can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the agents that modulate CIDE-A activity are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as efhylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the agent that modulates CIDE-A activity and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an agent for the treatment of subjects. Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Agents which exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such CIDE-A modulating agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the therapeutic methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 {i. e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g. , about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
The nucleic acid molecules used in the methods of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. C. Pharmacogenomics
In conjunction with the therapeutic methods of the invention, pharmacogenomics (i.e., the study of the relationship between a subject's genotype and that subject's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an agent which modulates CIDE-A activity, as well as tailoring the dosage and/or therapeutic regimen of treatment with an agent which modulates CIDE-A activity.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol. Physiol. 23(10-11): 983-985 andLinder, M.W. etal. (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate aminopeptidase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants). Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/TII drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease- associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals. Alternatively, a method termed the "candidate gene approach" can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug target is known (e.g., a CIDE-A protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and the cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6- formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Alternatively, a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a CIDE-A molecule or CIDE-A modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.
Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of a subject. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and, thus, enhance therapeutic or prophylactic efficiency when treating a subject suffering from a brown adipose cell disorder with an agent which modulates CIDE-A activity.
IV. Recombinant Expression Vectors and Host Cells Used in the Methods of the Invention
The methods of the invention (e.g., the screening assays described herein) include the use of vectors, preferably expression vectors, containing a nucleic acid encoding a CIDE-A protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors to be used in the methods of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CIDE-A proteins, mutant forms of CIDE-A proteins, fusion proteins, and the like). The recombinant expression vectors to be used in the methods of the invention can be designed for expression of CIDE-A proteins in prokaryotic or eukaryotic cells. For example, CIDE-A proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel (1990) supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a roteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Purified fusion proteins can be utilized in CIDE-A activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for CIDE-A proteins. In a preferred embodiment, a CIDE-a fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).
In another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
The methods of the invention may further use a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) ofan RNA molecule which is antisense to CIDE-A mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to the use of host cells into which a CIDE-A nucleic acid molecule of the invention is introduced, e.g., a CIDE-A nucleic acid molecule within a recombinant expression vector or a CIDE-A nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a CIDE-a protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. A host cell used in the methods of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce {i.e., express) a CIDE-A protein. Accordingly, the invention further provides methods for producing a CIDE-A protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a CIDE-A protein has been introduced) in a suitable medium such that a CIDE-A protein is produced. In another embodiment, the method further comprises isolating a CIDE-A protein from the medium or the host cell.
V. Isolated Nucleic Acid Molecules Used in the Methods of the Invention
The coding sequence of the isolated human CIDE-A cDNA and the predicted amino acid sequence of the human CIDE-A polypeptide are shown in SEQ ID NOs:l and 2, respectively. The CIDE-A sequence is also described in Inohara, et al. (1998), supra), the contents of which are incorporated herein by reference.
The methods of the invention include the use of isolated nucleic acid molecules that encode CIDE-A proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify CIDE-A-encoding nucleic acid molecules (e.g., CIDE-A mRNA) and fragments for use as PCR primers for the amplification or mutation of CIDE-A nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
A nucleic acid molecule used in the methods of the present invention, e.g. , a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:l as a hybridization probe, CIDE-A nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:l can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1.
A nucleic acid used in the methods of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Furthermore, oligonucleotides corresponding to CIDE-A nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
In a preferred embodiment, the isolated nucleic acid molecules used in the methods of the invention comprise the nucleotide sequence shown in SEQ ID NO:l, a complement of the nucleotide sequence shown in SEQ ID NO:l, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO: 1, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule used in the methods of the present invention comprises a nucleotide sequence which is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l or a portion of any of this nucleotide sequence.
Moreover, the nucleic acid molecules used in the methods of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a CIDE-A protein, e.g., a biologically active portion of a CIDE-A protein. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO: 1 of an anti-sense sequence of SEQ ID NO : 1 or of a naturally occurring allelic variant or mutant of SEQ ID NO: 1. In one embodiment, a nucleic acid molecule used in the methods of the present invention comprises a nucleotide sequence which is greater than 100, 100-200, 200-300, 300-400, 400-500, 500-600, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), chapters 7, 9 and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45° C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present invention. SSPE (lxSSPE is 0.15M NaCl, lOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5- 10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2{# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5 + 16.6(logιo[Na+]) + 0.41 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for IxSSC = 0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g. , EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2P04, 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH2P0 , 1% SDS at 65°C, see e.g., Church and Gilbert {1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2X SSC, 1% SDS). In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CIDE-A protein, such as by measuring a level of a CIDE-A-encoding nucleic acid in a sample of cells from a subject e.g., detecting CIDE-A mRNA levels or determining whether a genomic CIDE-A gene has been mutated or deleted.
The methods of the invention further encompass the use of nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l due to degeneracy of the genetic code and thus encode the same CIDE-A proteins as those encoded by the nucleotide sequence shown in SEQ ID NO: 1. In another embodiment, an isolated nucleic acid molecule included in the methods of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
The methods of the invention further include the use of allelic variants of human CIDE-A, e.g. , fuctional and non-functional allelic variants. Functional allelic variants are naturally occurring amino acid sequence variants of the human CIDE-A protein that maintain a CIDE-A activity. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally occurring amino acid sequence variants of the human CIDE-A protein that do not have a CIDE-A activity. Non-functional allelic variants will typically contain a non-conservative substitution, deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion or deletion in critical residues or critical regions of the protein. The methods of the present invention may further use non-human orthologues of the human CIDE-A protein. Orthologues of the human CIDE-A protein are proteins that are isolated from non-human organisms and possess the same CIDE-A activity.
The methods of the present invention further include the use of nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l or a portion thereof, in which a mutation has been introduced. The mutation may lead to amino acid substitutions at "non-essential" amino acid residues or at "essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CIDE-A (e.g., the sequence of SEQ ID NO: 2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the CIDE-A proteins of the present invention and other members of the CIDE family (e.g., CIDE-B, FSP-27, and DFF45) are not likely to be amenable to alteration.
Mutations can be introduced into SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains {e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains {e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a CIDE-A protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CIDE-A coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CIDE-A biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1 the encoded protein can be expressed recombinantly and the activity of the protein can be determined using the assay described herein.
Another aspect of the invention pertains to the use of isolated nucleic acid molecules which are antisense to the nucleotide sequence of SEQ ID NO: 1. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire CIDE-A coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a CIDE-A. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding CIDE-A. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding CIDE-A disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of CIDE-A mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of CIDE-A mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CIDE-A mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules used in the methods of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CIDE-A protein to thereby inhibit expression of the protein, e.g. , by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g. , by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule used in the methods of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue etal. (1987) FEBS Lett. 215:327-330). In still another embodiment, an antisense nucleic acid used in the methods of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CIDE-A mRNA transcripts to thereby inhibit translation of CIDE-A mRNA. A ribozyme having specificity for a CIDE-A-encoding nucleic acid can be designed based upon the nucleotide sequence of a CIDE-A cDNA disclosed herein (i.e., SEQ ID NO:l). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CIDE-A-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, CIDE-A mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
Alternatively, CIDE-A gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CIDE-A (e.g., the CIDE-A promoter and/or enhancers) to form triple helical structures that prevent transcription of the CIDE-A gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84; Helene, C. et al. (1992) Ann. N Y. Acad. Sci. 660:27-36; and Maher, L.J. {1992) Bioassays 14(12):807-15. '
In yet another embodiment, the CIDE-A nucleic acid molecules used in the methods of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93 : 14670-675.
PNAs of CIDE-A nucleic acid molecules can be used in the therapeutic and diagnostic applications described herein. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of CIDE-A nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) supra).
In another embodiment, PNAs of CIDE-A can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of CIDE-A nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g. , RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. et al. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. et al. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119- 11124). In other embodiments, the oligonucleotide used in the methods of the invention may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e. g. , PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent). VI. Isolated CIDE-A Proteins and Anti-CIDE-A Antibodies Used in the Methods of the Invention
The methods of the invention include the use of isolated CIDE-A proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-CIDE-A antibodies. In one embodiment, native CIDE-A proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, CIDE-A proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a CIDE-A protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
As used herein, a "biologically active portion" of a CIDE-A protein includes a fragment of a CIDE-A protein having a CIDE-A activity. Biologically active portions of a CIDE-A protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the CIDE-A protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include fewer amino acids than the full length CIDE-A proteins, and exhibit at least one activity of a CIDE-A protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the CIDE-A protein (e.g., the N-terminal region of the CIDE-A protein that is believed to be involved in the regulation of apoptotic activity). A biologically active portion of a CIDE-A protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300 or more amino acids in length. Biologically active portions of a CIDE-A protein can be used as targets for developing agents which modulate a CIDE-A activity. h a preferred embodiment, the CIDE-A protein used in the methods of the invention has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the CIDE-A protein is substantially identical to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection V above. Accordingly, in another embodiment, the CIDE-A protein used in the methods of the invention is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the CIDE-A amino acid sequence of SEQ ID NO:2 having 500 amino acid residues, at least 75, preferably at least 150, more preferably at least 225, even more preferably at least 300, and even more preferably at least 400 or more amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch {J. Mol Biol 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a-NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller {Comput. Appl Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The methods of the invention may also use CIDE-A chimeric or fusion proteins. As used herein, a CIDE-A "chimeric protein" or "fusion protein" comprises a CIDE-A polypeptide operatively linked to a non-CIDE-A polypeptide. An "CIDE-A polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a CIDE-A molecule, whereas a "non-CIDE-A polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the CIDE-A protein, e.g., a protein which is different from the CIDE-A protein and which is derived from the same or a different organism. Within a CIDE-A fusion protein the CIDE-A polypeptide can correspond to all or a portion of a CIDE-A protein. In a preferred embodiment, a CIDE-A fusion protein comprises at least one biologically active portion of a CIDE-A protein. In another preferred embodiment, a CIDE-A fusion protein comprises at least two biologically active portions of a CIDE-A protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the CIDE-A polypeptide and the non-CIDE-A polypeptide are fused in-frame to each other. The non-CIDE-A polypeptide can be fused to the N-terminus or C-terminus of the CIDE-A polypeptide. For example, in one embodiment, the fusion protein is a GST-CIDE-A fusion protein in which the CIDE-A sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant CIDE-A.
In another embodiment, this fusion protein is a CIDE-A protein containing a heterologous signal sequence at its N-tenninus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of CIDE-A can be increased through use of a heterologous signal sequence.
The CIDE-A fusion proteins used in the methods of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The CIDE-A fusion proteins can be used to affect the bioavailability of a CIDE-A substrate. Use of CIDE-A fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a CIDE-A protein; (ii) mis-regulation of the CIDE-A gene; and (iii) aberrant post-translational modification of a CIDE-A protein.
Moreover, the CIDE-A-fusion proteins used in the methods of the invention can be used as immunogens to produce anti-CIDE-A antibodies in a subject, to purify CIDE- A ligands and in screening assays to identify molecules which inhibit the interaction of CIDE-A with a CIDE-A substrate.
Preferably, a CIDE-A chimeric or fusion protein used in the methods of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CIDE-A-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CIDE-A protein.
The present invention also pertains to the use of variants of the CIDE-A proteins which function as either CIDE-A agonists (mimetics) or as CIDE-A antagonists. Variants of the CIDE-A proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a CIDE-A protein. An agonist of the CIDE-A proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a CIDE-A protein. An antagonist of a CIDE-A protein can inhibit one or more of the activities of the naturally occurring form of the CIDE-A protein by, for example, competitively modulating a CIDE-A-mediated activity of a CIDE-A protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the CIDE-A protein. In one embodiment, variants of a CIDE-A protein which function as either
CIDE-A agonists (mimetics) or as CIDE-A antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of a CIDE-A protein for CIDE-A protein agonist or antagonist activity. In one embodiment, a variegated library of CIDE-A variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of CIDE-A variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CIDE-A sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. , for phage display) containing the set of CIDE-A sequences therein. There are a variety of methods which can be used to produce libraries of potential
CIDE-A variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CIDE-A sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et α/. (1984) Annu. Rev. Biochem. 53:323; Itakura et α . (1984) Scz'ewce 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
In addition, libraries of fragments of a CIDE-A protein coding sequence can be used to generate a variegated population of CIDE-A fragments for screening and subsequent selection of variants of a CIDE-A protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CIDE-A coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the CIDE-A protein. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CIDE-A proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CIDE-A variants (Arkin and Yourvan (1992) Proc. Natl Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
The methods of the present invention further include the use of anti-CIDE-A antibodies. An isolated CIDE-A protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind CIDE-A using standard techniques for polyclonal and monoclonal antibody preparation. A full-length CIDE-A protein can be used or, alternatively, antigenic peptide fragments of CIDE-A can be used as immunogens. The antigenic peptide of CIDE-A comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of CIDE-A such that an antibody raised against the peptide forms a specific immune complex with the CIDE-A protein. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of CIDE-A that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
A CIDE-A immunogen is typically used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed CIDE-A protein or a chemically synthesized CIDE-A polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CIDE-A preparation induces a polyclonal anti-CIDE-A antibody response. The teπn "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as a CIDE-A. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind CIDE-A molecules. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CIDE-A. A monoclonal antibody composition thus typically displays a single binding affinity for a particular CIDE-A protein with which it immunoreacts.
Polyclonal anti-CIDE-A antibodies can be prepared as described above by immunizing a suitable subject with a CIDE-A immunogen. The anti-CIDE-A antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CIDE-A. If desired, the antibody molecules directed against CIDE-A can be isolated from the mammal (e.g. , from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-CIDE-A antibody titers are highest, antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.
(1976) Proc. Natl Acad. Sci. USA 76:2927-31; and Yeh et al (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Getter, M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a CIDE-A immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds CIDE-A. Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CIDE-A monoclonal antibody (see, e.g., G. Galfre et al (1977) Nature 266:55052; Gefter et al.
(1977) supra; Lerner (1981) supra; and Kenneth (1980) supra). Moreover, the ' ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind CIDE-A, e.g., using a standard ELISA assay. Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-CIDE-A antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CIDE-A to thereby isolate immunoglobulin library members that bind CIDE-A. Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275- 1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et «/. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554. Additionally, recombinant anti-CIDE-A antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the methods of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521 -3526; Sun et al (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et α/. (1987) Cane. Res. 47:999-1005; Wood et α/. (1985) Nature 314:446-449; S aw et al (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060.
An anti-CIDE-A antibody can be used to detect CIDE-A protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the CIDE-A protein. Anti-CIDE-A antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling {i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figure and the Sequence Listing is incorporated herein by reference.
EXAMPLES
EXAMPLE 1 : TISSUE DISTRIBUTION BY ANALYSIS OF CIDE-A AND FSP- 27 cDNA IN MOUSE TISSUES
This example describes the tissue distribution of CIDE-A and FSP-27 cDNA in normal mouse tissues. Tissues were collected from 7 week old female C57/B16 mice. RNA was prepared using the trizol method and treated with DNAse to remove contaminating genomic DNA. cDNA was synthesized using standard techniques. Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control GAPDH gene confirming efficient removal of genomic DNA contamination. The following primers (based on the mouse CIDE-A sequence) were used for Taqman™ (PE Applied Biosystems) analysis:
CIDE-A forward primer (SEQ ID NO:3): 5'CCAGCTCGCCCTTTTCG 3' CIDE-A reverse primer (SEQ ID NO:4): 5' TGCTGGCCATCACCCC 3' CIDE-1 probe (SEQ ID NO:5): 5' TCAAACCATGACCGAAGTAGCCGGC 3' FSP-27 forward primer (SEQ ID NO:6): 5' TCCTCCCAGCCTGTCTGAAG 3'
FSP-27 reverse primer (SEQ ID NO:7): 5' CATCCATGGCCCAAGGAAG 3' FSP-27 probe (SEQ ID NO:8): 5' TGCTGCAATGAAGACCCGAGTCCTC 3'. The Taqman ™ system was used according to the manufacturer's directions. To allow standardization between different tissues, each sample contained two probes distinguished by different fluorescent labels, a probe for the gene of interest {e.g., CIDE- A), as well as a probe for GAPDH as an internal control. The threshold values at which the PCR amplification started were determined using the manufacturer's software. PCR cycle number at threshold value was designated as CT. Relative expression was calculated as -((CTtest-CTGAPDH)tissue of interest - (CTtest-CTGAPDH)lowest expressing tissue in panel) c ,
— . ampies were run in duplicate and the averages of 2 relative expression determinations are shown in Figure 2A and 2B. All probes were tested on serial dilutions of RNA from a tissue with high expression levels and only probes which gave relative expression levels that were linear to the amount of template cDNA with a slope similar to the slope for the internal control GAPDH were used. As indicated in Figure 1 , the levels of CIDE-A detected in brown adipose tissue are approximately 20-fold higher compared to the levels of CIDE-A in white adipose tissue. Conversely, the levels of FSP-27 detected in white adipose tissue are approximately 10-fold higher compared to the levels of FSP-27 in brown adipose tissue.
EXAMPLE 2: IN SITU HYBRIDIZATION ANALYSIS OF MOUSE CIDE-
A
This example describes the use of in situ hybridization analysis to determine tissue distribution. For in situ analysis, various tissues, e.g. white and brown adipose tissues, were first frozen on dry ice. Ten-micrometer-thick sections of the tissues were postfixed with 4% formaldehyde in DEPC-treated IX phosphate-buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0J M triethanolamine-HCl (pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 M triethanolamine-HCl for 10 minutes, sections were rinsed in DEPC 2X SSC (IX SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissues were then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.
Hybridizations were performed with 35s-radiolabeled (5 X 10? cpm/ml) cRNA probes. Probes were incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01 % sheared salmon sperm DNA, 0.01 % yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55 °C.
After hybridization, slides were washed with 2X SSC. Sections were then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with 1 Oμg of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides were then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour. Sections were then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4°C for 7 days before being developed and counter stained.
In situ hybridization results show that the mouse CIDE-A gene is expressed at high levels in E15.5 mouse embryo and postnatal day 1.5 mouse with strong specific signal localized to brown adipose tissue. Also, there is high expression of CIDE-A in murine adult brown adipose tissue. There are low levels of expression of CIDE-A in testis and pancreas. There is no detected expression of CIDE-A in murine adult white adipose tissue, nor is there detected expression in murine adult brain, spleen, lymph node, spinal cord, ovary kidney, adrenal gland, or heart.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed:
1. A method for identifying a compound capable of treating a brown adipose cell disorder comprising assaying the ability of the compound to modulate CIDE-A nucleic acid expression or CIDE-A polypeptide activity, thereby identifying a compound capable of treating a brown adipose cell disorder.
2. The method of claim 1, wherein the brown adipose cell disorder is obesity.
3. The method of claim 1 , wherein the ability of the compound to modulate CIDE-A nucleic acid expression or CIDE-A polypeptide activity is determined by detecting apoptosis of a brown adipose cell.
4. The method of claim 1 , wherein the ability of the compound to modulate
CIDE-A nucleic acid expression or CIDE-A polypeptide activity is detennined by detecting increased thermogenesis in a brown adipose tissue.
5. A method for identifying a compound capable of modulating a brown adipose cell activity comprising: a) contacting a brown adipose cell with a test compound; and b) assaying the ability of the test compound to modulate the expression of a CIDE-A nucleic acid or. the activity of a CIDE-A polypeptide, thereby identifying a compound capable of modulating a brown adipose cell activity.
6. The method of claim 5, wherein said brown adipose cell activity is cell proliferation.
7. The method of claim 5, wherein said brown adipose cell activity is cell differentiation.
8. The method of claim 5, wherein said brown adipose cell activity is cell death.
9. A method for modulating a brown adipose cell activity comprising contacting a brown adipose cell with a CIDE-A modulator, thereby modulating said brown adipose cell activity.
10. The method of claim 9, wherein the CIDE-A modulator is a small molecule.
11. The method of claim 9, wherein said brown adipose cell activity is cell proliferation.
12. The method of claim 9, wherein said brown adipose cell activity is cell differentiation.
13. The method of claim 9, wherein said brown adipose cell activity is cell death.
14. The method of claim 9, wherein the CIDE-A modulator is capable of modulating CIDE-A polypeptide activity.
15. The method of claim 14, wherein the CIDE-A modulator is an anti- CIDE-A antibody.
16. The method of claim 14, wherein the CIDE-A modulator is a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof.
17. The method of claim 14, wherein the CIDE-A modulator is a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, wherein said percent identity is calculated using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
18. The method of claim 14, wherein the CIDE-A modulator is an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule consisting of SEQ ID NO:l at 6X SSC at 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
19. The method of claim 9, wherein the CIDE-A modulator is capable of modulating CIDE-A nucleic acid expression.
20. The method of claim 19, wherein the CIDE-A modulator is an antisense CIDE-A nucleic acid molecule.
21. The method of claim 19, wherein the CIDE-A modulator is a ribozyme.
22. The method of claim 19, wherein the CIDE-A modulator comprises the nucleotide sequence of SEQ ID NO:l, or a fragment thereof.
23. A method for treating a subject having a brown adipose cell disorder characterized by aberrant CIDE-A polypeptide activity or aberrant CIDE-A nucleic acid expression comprising administering to the subject a CIDE-A modulator, thereby treating said subject having a brown adipose cell disorder.
24. The method of claim 23, wherein said adipose cell disorder is obesity.
25. The method of claim 23, wherein said CIDE-A modulator is administered in a pharmaceutically acceptable formulation.
26. The method of claim 23, wherein said CIDE-A modulator is administered using a gene therapy vector.
27. The method of claim 23 , wherein the CIDE-A modulator is a small molecule.
28. The method of claim 23, wherein the CIDE-A modulator is capable of modulating CIDE-A polypeptide activity.
29. The method of claim 28, wherein the CIDE-A modulator is an anti- CIDE-A antibody.
30. The method of claim 28, wherein the CIDE-A modulator is a CIDE-A polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof.
31. The method of claim 28, wherein the CIDE-A modulator is a CIDE-A polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, wherein said percent identity is calculated using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
32. The method of claim 28, wherein the CIDE-A modulator is an isolated naturally occurring allelic variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule consisting of SEQ ID NO: 1 at 6X SSC at 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
33. The method of claim 23, wherein the CIDE-A modulator is capable of modulating CIDE-A nucleic acid expression.
34. The method of claim 33, wherein the CIDE-A modulator is an antisense CIDE-A nucleic acid molecule.
35. The method of claim 33, wherein the CIDE-A modulator is a ribozyme.
36. The method of claim 33, wherein the CIDE-A modulator comprises the nucleotide sequence of SEQ ID NO: 1 , or a fragment thereof.
37. A method for modulating thermogenesis in a subject comprising administering to the subject a CIDE-A modulator, thereby modulating thermogenesis in said subject.
38. A method for modulating apoptosis in a brown adipose cell in a subject comprising administering to the subject a CIDE-A modulator, thereby modulating apoptosis of a brown adipose cell in said subject.
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